Anticancer peptides

ABSTRACT

The present invention provides a peptide of formula (I) or a pharmaceutical salt thereof wherein “m”, “n”, “p”, and “q” represent integers and are selected from 0 and 1; and “r” is comprised from 1 to 10; a linker biradical of formula (II), which is connecting an alpha carbon atom of an amino acid located at position “i” in the peptide sequence of formula (I) with an alpha carbon atom of an amino acid located at position “i+4” or “i+7” in the peptide sequence of formula (I); a C-terminal end corresponding to —C(O)R 4 ; and a N-terminal end corresponding to —NHR 5 . The peptides of the invention show anticancer activity and an appropriate half-life and stability. Formula (I).

This application claims the benefit of European Patent ApplicationEP17382494.7 filed on Jul. 25, 2017.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 480386 402USPC SEQUENCE LISTING. The textfile is21.9 KB, was created on Nov. 5, 2021, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

This invention relates generally to the field of antineoplasticcompounds and, more particularly, to the design and synthesis ofpeptides with improved anticancer activity.

BACKGROUND ART

The therapeutic use of proteins and peptides that act intracellularlyholds much promise for the treatment of cancer and other diseases.

Cancer is the result in the occurrence of multiple factors. Mutationsmay occur in proto-oncogenes that cause cellular proliferation toincrease. Mutations also may occur in tumor suppressors whose normalfunction is to regulate cellular proliferation. Mutations in DNA repairenzymes impair the ability of the cell to repair damage beforeproliferating.

Tumor suppressor genes are normal genes whose absence (loss orinactivation) can lead to cancer. Tumor suppressor genes encode proteinsthat slow cell growth and division. Wild-type alleles of tumorsuppressor genes express proteins that suppress abnormal cellularproliferation. When the gene coding for a tumor suppressor protein ismutated or deleted, the resulting mutant protein or the complete lack oftumor suppressor protein expression may fail to correctly regulatecellular proliferation, and abnormal cellular proliferation may takeplace, particularly if there is already existing damage to the cellularregulatory mechanism. A number of well-studied human tumors and tumorcell lines have been shown to have missing or nonfunctional tumorsuppressor genes.

Currently, there are few effective options for the treatment of manycommon cancer types. The course of treatment for a given individualdepends on the diagnosis, the stage to which the disease has developedand factors such as age, sex and general health of the patient. The mostconventional options of cancer treatment are surgery, radiation therapyand chemotherapy. These therapies each are accompanied with varying sideeffects and they have varying degrees of efficacy.

These side effects, together with the multidrug resistance alreadydisclosed for traditional chemotherapy, have prompted urgent needs fornovel anticancer drugs or therapeutic approaches.

Anticancer peptides have become promising molecules for novel anticanceragents because of their unique mechanism and several extraordinaryproperties. However, properties such as the specificity and sensitivityshown by the peptides already disclosed in the prior art, need furtherimprovement.

Thus, in spite of the efforts made, there is still the need ofdeveloping further polypeptides with appropriate anticancer profile.

SUMMARY OF INVENTION

The present inventors found that sequence SEQ ID NO: 1 was not effectivein inhibiting cancer cell proliferation. Surprisingly, when saidsequence SEQ ID NO: 1 was modified, either by the inclusion of a sidechain bridge (stapling) or by conjugation to a cell penetrating peptide,the peptide sequence became remarkably active in inhibiting cancer cellproliferation (see Tables 5-6 below). The efficiency showed by thepeptides of the invention makes that a small amount is required toachieve the desired effect.

It is known in the state of the art that hepatotoxicity is a side-effectrelated to the administration of anticancer compounds. As it is shown inTable 7 below, the peptides of the invention show such anticancer effectwithout giving rise to toxicity in human hepatocytes.

Thus, in a first aspect the present invention provides an anticancerpeptide of formula (I) or a pharmaceutical salt thereof:

wherein

“m”, “n”, “p”, and “q” represent integers and are selected from 0 and 1;and

“r” is comprised from 1 to 10;

the peptide comprising:

-   -   a linker biradical “L” of formula (II)        —[(R₁)_(a)—(R₂)—(R₃)_(b)]_(c)—  (II)

which is connecting an alpha carbon atom of an amino acid located atposition “i” in the peptide of formula (I) with an alpha carbon atom ofan amino acid located at position “i+4” or “i+7” in the peptide offormula (I),

-   -   a C-terminal end corresponding to —C(O)R₄; and    -   a N-terminal end corresponding to —NHR₅;

wherein

-   -   “a” and “b” are the same or different and are 0 or 1;    -   “c” is comprised from 1 to 10;    -   R₁ and R₃ are biradicals independently selected from the group        consisting of: (C₁-C₁₀)alkyl; (C₁-C₁₀)alkyl substituted by one        or more radicals selected from the group consisting of: halogen,        (C₁-C₁₀)alkyl, —OR₆, —NR₇R₈, —SR₉, —SOR₁₀, —SO₂R₁₁, and —CO₂R₁₂;        (C₂-C₁₀)alkenyl; (C₂-C₁₀)alkenyl substituted by one or more        radicals selected from the group consisting of: halogen,        (C₁-C₁₀)alkyl, —OR₆, —NR₇R₈, —SR₉, —SOR₁₀, —SO₂R₁₁, and —CO₂R₁₂;        (C₂-C₁₀)alkynyl; and (C₂-C₁₀)alkinyl substituted by one or more        radicals selected from the group consisting of: halogen,        (C₁-C₁₀)alkyl, —OR₆, —NR₇R₈, —SR₉, —SOR₁₀, —SO₂R₁₁, and —CO₂R₁₂;    -   R₂ is a biradical selected from the group consisting of: —O—,        C(═O), C(═O)NR₁₃, C(═O)O, S(═O), S(═O)₂, NR₁₄, (C₁-C₁₀)alkyl,        (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl, —NR₁₅—NR₁₆—, —N═N—, —S—S—, and        a known ring system comprising from 3 to 14 members, the system        comprising from 1 to 3 rings, where:        -   each one of the rings is saturated, partially unsaturated,            or aromatic;        -   the rings are isolated, partially or totally fused,        -   each one of the members forming the known ring system is            selected from the group consisting of: —CH—, —CH₂—, —NH—,            —N—, —SH—, —S—, and —O—; and        -   the ring system is optionally substituted by one or more            radicals independently selected from the group consisting of            halogen, —OH, —NO₂, (C₁-C₁₀)alkyl, (C₁-C₁₀)haloalkyl, and            (C₁-C₁₀)alkyl-O—; and    -   R₄ is a radical selected from the group consisting of —OH and        —NR₁₇R₁₈;    -   R₅ is a radical selected from the group consisting of —H and        (C₁-C₂₀)alkyl;    -   R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ R₁₃, R₁₄, R₁₅, R₁₆, R₁₇ and R₁₈        are radicals independently selected from the group consisting        of: —H and (C₁-C₁₀)alkyl; and    -   the amino acids, which are connected by the linker, being of        formula (III)

-   -   wherein        -   R₁₉ is a monoradical selected from the group consisting of:            —H, (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl, and a            known ring system comprising from 3 to 14 members, the            system comprising from 1 to 3 rings, where:        -   each one of the rings is saturated, partially unsaturated,            or aromatic;        -   the rings are isolated, partially or totally fused,        -   each one of the members forming the known ring system is            selected from the group consisting of: —CH—, —CH₂—, —NH—,            —N—, —SH—, —S—, and —O—;

or, alternatively, a peptide or pharmaceutical acceptable salt thereofhaving an identity of at least 85% with the peptide of sequence SEQ IDNo: 18,

wherein

-   -   L is a linker biradical of formula (II) as defined above, and    -   X is an amino acid of formula (III), as defined above.

Due to such cancer cell proliferative inhibition, the peptides of theinvention are promising anticancer peptides.

In a second aspect, the present invention provides peptide having anidentity with respect to sequence SEQ ID NO:1 of at least 85%, or apharmaceutical salt thereof, conjugated to a cell penetrating peptide.

In addition to the above, as it is provided below, the peptides of theinvention show an appropriate half-life and stability in plasma, theamount (and effect) of the peptide being sustained in time for about 24hours after its incubation in human plasma (Table 8, below).

These data allow concluding that the peptides of the invention aresuitable as anti-cancer therapeutics.

In a third aspect the present invention provides a fusion proteincomprising the peptide as defined in the first or second aspect of theinvention.

In a fourth aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of the peptideor pharmaceutical salt thereof of the invention, or the fusion proteinas defined in the third aspect of the invention, together withacceptable pharmaceutical excipients and/or carriers.

In a fifth aspect, the present invention provides the peptide orpharmaceutical salt thereof as defined in the first or second aspect ofthe invention or the fusion protein as defined in the third aspect ofthe invention for use as a medicament. This aspect can be alternativelyformulated as the use of a peptide or pharmaceutical salt thereof asdefined in the first or second aspect of the invention or the fusionprotein as defined in the third aspect of the invention, in themanufacture of a medicament for the treatment of a disease. This aspectcan also be alternatively formulated as a method for the treatment of adisease, the method comprising administering an effective therapeuticamount of a peptide or pharmaceutical salt thereof as defined in thefirst or second aspect of the invention or of the fusion protein of thethird aspect of the invention, to a subject in need thereof.

In a sixth aspect, the present invention provides the peptide orpharmaceutical salt thereof as defined in the first or second aspect ofthe invention or the fusion protein as defined in the third aspect ofthe invention or the pharmaceutical composition as defined in the fourthaspect of the invention for use in the treatment of cancer. This aspectcan alternatively be formulated as the use of the peptide orpharmaceutical salt thereof as defined in the first or second aspect ofthe invention or the fusion protein as defined in the third aspect ofthe invention or the pharmaceutical composition as defined in the fourthaspect of the invention in the manufacture of a medicament for thetreatment of cancer. This aspect can also be alternatively formulated asa method for the treatment of cancer, the method comprisingadministering an effective therapeutic amount of the peptide orpharmaceutical salt thereof as defined in the first or second aspect ofthe invention or the fusion protein as defined in the third aspect ofthe invention or the pharmaceutical composition as defined in the fourthaspect of the invention, to a subject in need thereof.

In further aspects the present invention provides: (a) a combinationcomprising the peptide or pharmaceutical salt thereof as defined in thefirst or second aspect of the invention, or the fusion peptide asdefined in the third aspect of the invention or the pharmaceuticalcomposition of the fourth aspect of the invention; and a therapeuticagent, particularly an anti-cancer agent; (b) a combination comprisingthe peptide or pharmaceutical salt thereof as defined in the first orsecond aspect of the invention, or the fusion peptide of the thirdaspect of the invention or the pharmaceutical composition of the fourthaspect of the invention; and a therapeutic agent, particularly ananti-cancer agent, for use as a medicament, more particularly for use inthe treatment of cancer; (c) a peptide or pharmaceutical salt thereof asdefined in the first or second aspect of the invention, or the fusionpeptide as defined in the third aspect of the invention, or thepharmaceutical composition as defined in the fourth aspect of theinvention for use in combination therapy for the prevention or treatmentof cancer, wherein the therapy comprises its administration to a subjectsimultaneously, sequentially or separately with an anti-cancer agent;and (d) an anti-cancer agent for use in combination therapy with thepeptide or pharmaceutical salt thereof as defined in the first or secondaspect of the invention, or the fusion peptide as defined in the thirdaspect of the invention, or the pharmaceutical composition as defined inthe fourth aspect of the invention, wherein the use comprises theprevention or treatment of cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a Western-blot analysis of the amount of cleaved Parp proteininduced by the administration of H14 peptide of the invention.

FIG. 2 is a Western-blot analysis of the amount of Caspase 3 induced bythe administration of H14 peptide of the invention.

DETAILED DESCRIPTION OF THE INVENTION

All terms as used herein in this application, unless otherwise stated,shall be understood in their ordinary meaning as known in the art. Othermore specific definitions for certain terms as used in the presentapplication are as set forth below and are intended to apply uniformlythrough-out the specification and claims unless an otherwise expresslyset out definition provides a broader definition.

For the purposes of the present invention, any ranges given include boththe lower and the upper end-points of the range.

The present invention provides peptides comprising sequences of formula(I) or alternatively having an identity of at least 85% with the peptideof SEQ ID NO: 18, as it has been stated above.

As used herein, the term “pharmaceutical acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticalacceptable salts are well known in the art. Examples of pharmaceuticallyacceptable, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, trifluoroacetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Other pharmaceuticalacceptable salts include adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, and ammonium. Representative alkali or alkalineearth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutical acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

The term (C₁-C₁₀)alkyl refers to a saturated straight or branched alkylchain having from 1 to 10 carbon atoms. Illustrative non-limitativeexamples are: methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, neo-pentyl and n-hexyl.

The term (C₁-C₂₀)alkyl refers to a saturated straight or branched alkylchain having from 1 to 20 carbon atoms.

The term (C₂-C₁₀)alkenyl refers to a saturated straight, or branchedalkyl chain containing from 2 to carbon atoms and also containing one ormore double bonds. Illustrative non-limitative examples are ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

The term (C₂-C₁₀)alkynyl refers to a saturated straight, or branchedalkyl chain containing from 2 to carbon atoms and also containing one ormore triple bonds. Examples include, among others, ethynyl, 1-propynyl,2-butynyl, 1,3-butadinyl, 4-pentynyl, and 1-hexynyl.

The term “halogen” refers to the group in the periodic table consistingof five chemically related elements: fluorine (F), chlorine (Cl),bromine (Br), iodine (I), and astatine (At).

The term (C₁-C₁₀)haloalkyl refers to a group resulting from thereplacement of one or more hydrogen atoms from a (C₁-C₁₀)alkyl groupwith one or more, preferably from 1 to 6, halogen atoms, which can bethe same or different. Examples include, among others, trifluoromethyl,fluoromethyl, 1-chloroethyl, 2-chloroethyl, 1-fluoroethyl,2-fluoroethyl, 2-bromoethyl, 2-iodoethyl, 2,2,2-trifluoroethyl,pentafluoroethyl, 3-fluoropropyl, 3-chloropropyl,2,2,3,3-tetrafluoropropyl, 2,2,3,3,3-pentafluoropropyl,heptafluoropropyl, 4-fluorobutyl, and nonafluorobutyl.

The term “known” ring system as used herein refers to a ring systemwhich is chemically feasible and is known in the art and so intends toexclude those ring systems that are not chemically possible.

According to the present invention when the ring system is formed by“isolated” rings means that the ring system is formed by two, three orfour rings and said rings are bound via a bond from the atom of one ringto the atom of the other ring. The term “isolated” also embraces theembodiment in which the ring system has only one ring. Illustrativenon-limitative examples of known ring systems consisting of one ring arethose derived from: cyclopropyl, cyclobutyl, cyclopentyl, cyclhexyl,cycloheptyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, phenyl, andcycloheptenyl.

According to the present invention when the ring system has rings“totally fused”, means that the ring system is formed by two, three orfour rings in which two or more atoms are common to two adjoining rings.Illustrative non-limitative examples are 1,2,3,4-tetrahydronaphthyl,1-naphthyl, 2-naphthyl, anthryl, or phenanthryl.

According to the present invention when the ring system is “partiallyfused” it means that the ring system is formed by three or four rings,being at least two of said rings totally fused (i.e. two or more atomsbeing common to the two adjoining rings) and the remaining ring(s) beingbound via a bond from the atom of one ring to the atom of one of thefused rings.

The term “biradical” as used in the present application, means aneven-electron chemical compound with two free radical centres which actindependently of each other (IUPAC Gold Book, web page:http://goldbook.iupac.org, or IUPAC book, Version 2.3.3, 2014 Feb. 24,page 168).

Unless otherwise stated, the amino acids forming the peptides of theinvention can have L- or D-configuration.

Amino acids used in the construction of peptides of the presentinvention may be prepared by organic synthesis, or obtained by otherroutes, such as, for example, degradation of or isolation from a naturalsource.

In one embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided below, when the peptideis of formula (I), r is from 1 to 3. In another embodiment of the firstaspect of the invention, optionally in combination with any of theembodiments provided below, when the peptide is of formula (I), r is 1.Thus, the peptide of the first aspect of the invention consists of theamino acid sequence of formula (I)

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, a=1.In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided below, b=1. Inanother embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, c=1. Inanother embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below,a=b=c=1. In another embodiment of the first aspect of the invention,optionally in combination with any of the embodiments provided above orbelow, a=b=c=r=1.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, R₁and R₃ are biradicals independently selected from the group consistingof: (C₁-C₁₀)alkyl; (C₂-C₁₀)alkenyl; and (C₂-C₁₀)alkynyl. In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, R₁ andR₃ are the same or different and represent (C₁-C₁₀)alkyl.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, R₂is a biradical selected from the group consisting: —O—, C(═O),C(═O)NR₁₃, C(═O)O, S(═O), S(═O)₂, NR₁₄, (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl,(C₂-C₁₀)alkynyl, —NR₁₅—NR₁₆—, —N═N—, —S—S—, and a known ring systemconsisting of one ring from 3 to 6 members, the ring:

-   -   being saturated, partially unsaturated, or aromatic;    -   each one of the members forming the known ring system being        selected from the group consisting of: —CH—, —CH₂—, —NH—, —N—,        —SH—, —S—, and —O—; and    -   the ring system being optionally substituted by one or more        radicals independently selected from the group consisting of        halogen, —OH, —NO₂, (C₁-C₁₀)alkyl, (C₁-C₁₀)haloalkyl, and        (C₁-C₁₀)alkyl-O—.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, R₂is a biradical selected from the group consisting of: (C₁-C₁₀)alkyl,(C₂-C₁₀)alkenyl, and (C₂-C₁₀)alkynyl. In another embodiment of the firstaspect of the invention, optionally in combination with any of theembodiments provided above or below, R₂ is (C₂-C₁₀)alkenyl.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, R₁and R₃ are the same or different and represent (C₁-C₁₀)alkyl; and R₂ is(C₂-C₁₀)alkenyl.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, R₁and R₃ are the same or different and represent (C₁-C₁₀)alkyl; R₂ is(C₂-C₁₀)alkenyl; and a=b=c=1.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, R₁and R₃ are the same or different and represent (C₁-C₁₀)alkyl; R₂ is(C₂-C₁₀)alkenyl; and r=a=b=c=1.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, R₁₉is selected from the group consisting of: (C₁-C₁₀)alkyl,(C₂-C₁₀)alkenyl, and (C₂-C₁₀)alkynyl. In another embodiment of the firstaspect of the invention, optionally in combination with any of theembodiments provided above or below, R₁₉ is a (C₁-C₁₀)alkyl monoradical.In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, R₁,R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl; and R₂is (C₂-C₁₀)alkenyl.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, R₄is —OH (i.e., the C-terminal end is —C(O)OH). In another embodiment ofthe first aspect of the invention, optionally in combination with any ofthe embodiments provided above or below, R₄ is —NR₁₇R₁₈, R₁₇ and R₁₈having the same meaning. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, R₄ is —NH₂ (i.e., the C-terminal end is—C(O)NH₂).

In another embodiment of the first aspect of the invention, optionallyin combination with anty of the embodiments provided above or below, theN-terminal end corresponds to —NH₂. In another embodiment of the firstaspect of the invention, optionally in combination with any of theembodiments provided above or below, the C-terminal and N-terminal endsof the peptide of the invention are, respectively, —C(O)OH and —NH₂. Inanother embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, theC-terminal and N-terminal ends of the peptide of the invention are,respectively, —C(O)NH₂. and —NH₂.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, “m”and “n” means the same (i.e., both are 0 or 1).

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, “p”and “q” means the same (i.e., both are 0 or 1).

In one embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt corresponds to one of formula (I):

wherein

“m”, “n”, “p”, “q”, and “r” are as defined above,

and the peptide comprises a linker biradical of formula (II), aC-terminal end and a N-terminal end as defined in any of the embodimentsprovided above.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, thepeptide of formula (I) or a pharmaceutical salt thereof is one offormula (Ia) or (Ib):

wherein “m”, “n”, “p”, L, and R₁₉ are as defined above.

In one embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ia). Inanother embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ia), wherein“m” and “n” are the same. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ia) wherein “m” and “n” are 1. In another embodiment ofthe first aspect of the invention, optionally in combination with any ofthe embodiments provided above or below, the peptide or pharmaceuticalsalt thereof is one of formula (Ia) wherein “p” and “q” are 0. Inanother embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ia), whereinR₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl;and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ia) wherein “m” and “n” are 1, “p” and “q” are 0, R₁, R₃and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl; and R₂ is(C₂-C₁₀)alkenyl. In another embodiment of the first aspect of theinvention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ia) wherein “m” and “n” are 1, “p” and “q” are 0,a=b=c=1, R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ia) wherein “m” and “n” are 1, “p” and “q”are 0, r=a=b=c=1, R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl.

In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ib). Inanother embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ib), wherein“m” and “n” are the same. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ib) wherein “p” and “q” are the same. In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ib), whereinR₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl;and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ia) wherein “m” and “n” are 1, “p” and “q” are 0, R₁, R₃and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl; and R₂ is(C₂-C₁₀)alkenyl. In another embodiment of the first aspect of theinvention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ib) wherein “m” and “n” are 0, “p” and “q” are 1, R₁, R₃and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl; and R₂ is(C₂-C₁₀)alkenyl. In another embodiment of the first aspect of theinvention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ib) wherein “m” and “n” are 1, “p” and “q” are 0,a=b=c=1, R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ib) wherein “m” and “n” are 0, “p” and “q”are 1, a=b=c=1, R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ib) wherein “m” and “n” are 0, “p” and “q”are 1, r=a=b=c=1, R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl.

Alternatively, in another embodiment of the first aspect of theinvention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone having at least a 85% of identity with the sequence SEQ ID NO: 18.In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, thepeptide has an identity with respect of sequence SEQ ID NO: 18 of 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof, which has at least a 85% ofidentity with the sequence SEQ ID NO: 18, is selected from the groupconsisting of: (Ibis1), (Ibis2), (Ibis3) (Ibis4), (Ibis5) and (Ibis6):

being AA₃ an amino acid other than Thr, and “X” and “L” as defined inany of the above embodiments;

being AA₄ other than Glu, and “X” and “L” as defined in any of the aboveembodiments;

being AA₅ other than Pro, and “X” and “L” as defined in any of the aboveembodiments;

being AA₂ other than Ala, AA₃ other than Thr, AA₄ other than Glu, and“X” and “L” as defined in any of the above embodiments;

being AA₁ other than Asn, and “X” and “L” as defined in any of the aboveembodiments;

being AA₂ other than Ala, and “X” and “L” as defined in any of the aboveembodiments.

In another embodiment of the first aspect of the invention, the peptideor pharmaceutical salt corresponds to one of formula (Ibis1). In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis1),wherein R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis1) wherein a=b=c=1. In another embodimentof the first aspect of the invention, optionally in combination with anyof the embodiments provided above or below, the peptide orpharmaceutical salt thereof is one of formula (Ibis1) wherein a=b=c=1,R₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl;and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis1), AA₃ is a non-polar amino acid. In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis1) whereinAA₃ is Ala. In another embodiment of the first aspect of the invention,optionally in combination with any of the embodiments provided above orbelow, the peptide or pharmaceutical salt thereof is one of formula(Ibis1) wherein R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; and AA₃ is a non-polar amino acid.In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis1) whereina=b=c=1, R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; and AA₃ is a non-polar amino acid.In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis1) whereinR₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl; R₂is (C₂-C₁₀)alkenyl; and AA₃ is Ala. In another embodiment of the firstaspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis1) wherein “m” and “n” a=b=c=1, R₁, R₃and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl; R₂ is(C₂-C₁₀)alkenyl; and AA₃ is Ala.

In another embodiment of the first aspect of the invention, the peptideor pharmaceutical salt corresponds to one of formula (Ibis2). In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis2),wherein R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis2) wherein a=b=c=1. In another embodimentof the first aspect of the invention, optionally in combination with anyof the embodiments provided above or below, the peptide orpharmaceutical salt thereof is one of formula (Ibis2) wherein a=b=c=1,R₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl;and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis2), AA₄ is a non-polar amino acid or polar aminoacid. In another embodiment of the first aspect of the invention,optionally in combination with any of the embodiments provided above orbelow, the peptide or pharmaceutical salt thereof is one of formula(Ibis2) wherein AA₄ is Ala, Gln or Phe. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis2) wherein R₁, R₃ and R₁₉ are the same ordifferent and represent (C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; and AA₄ isa non-polar amino acid or polar amino acid. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis2) wherein a=b=c=1, R₁, R₃ and R₁₉ arethe same or different and represent (C₁-C₁₀)alkyl; R₂ is(C₂-C₁₀)alkenyl; and AA₄ is a non-polar amino acid or polar amino acid.In another embodiment of the first aspect of the invention, optionallyin combination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis2) whereinR₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl; R₂is (C₂-C₁₀)alkenyl; and AA₄ is Ala, Phe or Gln. In another embodiment ofthe first aspect of the invention, optionally in combination with any ofthe embodiments provided above or below, the peptide or pharmaceuticalsalt thereof is one of formula (Ibis2) wherein a=b=c=1, R₁, R₃ and R₁₉are the same or different and represent (C₁-C₁₀)alkyl; R₂ is(C₂-C₁₀)alkenyl; and AA₄ is Ala, Phe or Gln.

In another embodiment of the first aspect of the invention, the peptideor pharmaceutical salt corresponds to one of formula (Ibis3). In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis3),wherein R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis3) wherein a=b=c=1. In another embodimentof the first aspect of the invention, optionally in combination with anyof the embodiments provided above or below, the peptide orpharmaceutical salt thereof is one of formula (Ibis3) wherein a=b=c=1,R₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl;and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis3), AA₅ is a polar amino acid or Phe. In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis3) whereinAA₅ is Ser, Gln or Phe. In another embodiment of the first aspect of theinvention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis3) wherein R₁, R₃ and R₁₉ are the same or differentand represent (C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; and AA₅ is a polaramino acid or Phe. In another embodiment of the first aspect of theinvention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis3) wherein a=b=c=1, R₁, R₃ and R₁₉ are the same ordifferent and represent (C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; and AA₅ isa polar amino acid or Phe. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis3) wherein R₁, R₃ and R₁₉ are the same or differentand represent (C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; and AA₅ is Ser, Glnor Phe. In another embodiment of the first aspect of the invention,optionally in combination with any of the embodiments provided above orbelow, the peptide or pharmaceutical salt thereof is one of formula(Ibis3) wherein a=b=c=1, R₁, R₃ and R₁₉ are the same or different andrepresent (C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; and AA₅ is Ser, Gln orPhe.

In another embodiment of the first aspect of the invention, the peptideor pharmaceutical salt corresponds to one of formula (Ibis4). In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis4),wherein R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis4) wherein a=b=c=1. In another embodimentof the first aspect of the invention, optionally in combination with anyof the embodiments provided above or below, the peptide orpharmaceutical salt thereof is one of formula (Ibis4) wherein a=b=c=1,R₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl;and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis4), AA₂ is a polar amino acid, AA₃ is a non-polaramino acid, and AA₄ is a polar amino acid. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis4) wherein AA₂ is Gln. In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis4) whereinAA₃ is Ala. In another embodiment of the first aspect of the invention,optionally in combination with any of the embodiments provided above orbelow, the peptide or pharmaceutical salt thereof is one of formula(Ibis4) wherein AA₄ is Gln. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis4) wherein R₁, R₃ and R₁ are the same or differentand represent (C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; AA₂ is a polar aminoacid, AA₃ is a non-polar amino acid, and AA₄ is a polar amino. Inanother embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis4) whereina=b=c=1, R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; AA₂ is a polar amino acid, AA₃ isa non-polar amino acid, and AA₄ is a polar amino acid. In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis4) whereinR₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl; R₂is (C₂-C₁₀)alkenyl; AA₂ is Gln; AA₃ is Ala; and AA₄ is Gln. In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis4) whereina=b=c=1, R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; AA₂ is Gln; AA₃ is Ala; and AA₄ isGln.

In another embodiment of the first aspect of the invention, the peptideor pharmaceutical salt corresponds to one of formula (Ibis5). In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis5),wherein R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis5) wherein a=b=c=1. In another embodimentof the first aspect of the invention, optionally in combination with anyof the embodiments provided above or below, the peptide orpharmaceutical salt thereof is one of formula (Ibis5) wherein a=b=c=1,R₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl;and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis5), AA₁ is a polar amino acid other than Asn. Inanother embodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis5), AA₁ isGln. In another embodiment of the first aspect of the invention,optionally in combination with any of the embodiments provided above orbelow, the peptide or pharmaceutical salt thereof is one of formula(Ibis5) wherein R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; and AA₁ is a polar amino acidother than Asn. In another embodiment of the first aspect of theinvention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis5) wherein a=b=c=1, R₁, R₃ and R₁₉ are the same ordifferent and represent (C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; and AA₁ isGln.

In another embodiment of the first aspect of the invention, the peptideor pharmaceutical salt corresponds to one of formula (Ibis6). In anotherembodiment of the first aspect of the invention, optionally incombination with any of the embodiments provided above or below, thepeptide or pharmaceutical salt thereof is one of formula (Ibis6),wherein R₁, R₃ and R₁₉ are the same or different and represent(C₁-C₁₀)alkyl; and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of thefirst aspect of the invention, optionally in combination with any of theembodiments provided above or below, the peptide or pharmaceutical saltthereof is one of formula (Ibis6) wherein a=b=c=1. In another embodimentof the first aspect of the invention, optionally in combination with anyof the embodiments provided above or below, the peptide orpharmaceutical salt thereof is one of formula (Ibis6) wherein a=b=c=1,R₁, R₃ and R₁₉ are the same or different and represent (C₁-C₁₀)alkyl;and R₂ is (C₂-C₁₀)alkenyl. In another embodiment of the first aspect ofthe invention, optionally in combination with any of the embodimentsprovided above or below, the peptide or pharmaceutical salt thereof isone of formula (Ibis6), AA₂ is a polar amino acid. In another embodimentof the first aspect of the invention, optionally in combination with anyof the embodiments provided above or below, the peptide orpharmaceutical salt thereof is one of formula (Ibis6) wherein R₁, R₃ andR₁₉ are the same or different and represent (C₁-C₁₀)alkyl; R₂ is(C₂-C₁₀)alkenyl; and AA₂ is a polar amino acid. In another embodiment ofthe first aspect of the invention, optionally in combination with any ofthe embodiments provided above or below, the peptide or pharmaceuticalsalt thereof is one of formula (Ibis6) wherein a=b=c=1, R₁, R₃ and R₁₉are the same or different and represent (C₁-C₁₀)alkyl; R₂ is(C₂-C₁₀)alkenyl; and AA₂ is Gln.

In the present invention, by “non-polar amino acid” it is understood anyamino acid with hydrophobic nature. Non-polar amino acids have sidechains which are hydrocarbon alkyl groups (alkane branches) or aromatic(benzene rings) or heteroaromatic (e.g. indole ring). Illustrativenon-limitative examples of common non-polar amino acids are Ala, Val,Leu, Ile, Pro, Trp, Gly, Phe, and Met.

In the present invention, by “polar amino acid” it is to be understoodany polar-neutral amino acid, which has polar but not charged groups atneutral pH in the side chain (such as hydroxyl, amide or thiol groups).Illustrative non-limitative examples of polar neutral amino acids areSer, Thr, Cys, Tyr, Asn, and Gln.

In another embodiment of the invention, the peptide is selected from thegroup consisting of sequence having at least 85% of sequence identitywith any of the peptide sequences SEQ ID NO: 2 to 14. In anotherembodiment of the invention, the peptide is selected from the groupconsisting of sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity with anyof the peptide sequences SEQ ID NO: 2 to 14. In another embodiment ofthe invention, the peptide is selected from the group consisting ofsequence SEQ ID NO: 2 to 14. In another embodiment of the invention, thepeptide is selected from the group consisting of sequence SEQ ID NO: 2to 4.

In a second aspect the present invention provides a peptide having atleast an identity of sequence with respect to sequence SEQ ID NO:1 of atleast 85%, or a pharmaceutical salt thereof, conjugated to a cellpenetrating peptide.

In one embodiment of the second aspect of the invention, the peptide hasan identity with respect of sequence SEQ ID NO: 1 of 90, 91, 92, 93, 94,95, 96, 97, 98, 99 or 100%.

In the present invention the term “identity” refers to the percentage ofresidues or bases that are identical in the two sequences when thesequences are optimally aligned. If, in the optimal alignment, aposition in a first sequence is occupied by the same amino acid residueor nucleotide as the corresponding position in the second sequence, thesequences exhibit identity with respect to that position. The level ofidentity between two sequences (or “percent sequence identity”) ismeasured as a ratio of the number of identical positions shared by thesequences with respect to the size of the sequences (i.e., percentsequence identity=(number of identical positions/total number ofpositions)×100). In one embodiment, when the identity is calculated withrespect to a peptide of sequence SEQ ID NO: 18, the two aminoacidicresidues connected by the linker, are not considered when performing thealignment to determine the identical positions.

A number of mathematical algorithms for rapidly obtaining the optimalalignment and calculating identity between two or more sequences areknown and incorporated into a number of available software programs.Examples of such programs include the MATCH-BOX, MULTAIN, GCG, FASTA,and ROBUST programs for amino acid sequence analysis, among others.Preferred software analysis programs include the ALIGN, CLUSTAL W, andBLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof).

For amino acid sequence analysis, a weight matrix, such as the BLOSUMmatrixes (e.g., the BLOSUM45, BLOSUM50, BLOSUM62, and BLOSUM80matrixes), Gonnet matrixes, or PAM matrixes (e.g., the PAM30, PAM70,PAM120, PAM160, PAM250, and PAM350 matrixes), are used in determiningidentity.

The BLAST programs provide analysis of at least two amino acidsequences, either by aligning a selected sequence against multiplesequences in a database (e.g., GenSeq), or, with BL2SEQ, between twoselected sequences. BLAST programs are preferably modified by lowcomplexity filtering programs such as the DUST or SEG programs, whichare preferably integrated into the BLAST program operations. If gapexistence costs (or gap scores) are used, the gap existence costpreferably is set between about −5 and −15. Similar gap parameters canbe used with other programs as appropriate. The BLAST programs andprinciples underlying them are further described in, e.g., Altschul etal., “Basic local alignment search tool”, 1990, J. Mol. Biol, v. 215,pages 403-410.

For multiple sequence analysis, the CLUSTAL W program can be used. TheCLUSTAL W program desirably is run using “dynamic” (versus “fast”)settings. Amino acid sequences are evaluated using a variable set ofBLOSUM matrixes depending on the level of identity between thesequences. The CLUSTAL W program and underlying principles of operationare further described in, e.g., Higgins et al., “CLUSTAL V: improvedsoftware for multiple sequence alignment”, 1992, CABIOS, 8(2), pages189-191.

In the present invention the term “cell penetrating peptide” (“CPP”)refers to short peptides that facilitate cellular uptake of variousmolecular cargo (from nanosize particles to small chemical molecules andlarge fragments of DNA). The “cargo” is associated to peptides via theC(t) or N(t)-end, either through chemical linkage via covalent bonds orthrough non-covalent interactions. The function of the CPPs are todeliver the cargo into cells, a process that commonly occurs throughendocytosis with the cargo delivered to delivery vectors for use inresearch and medicine. Current use is limited by a lack of cellspecificity in CPP-mediated cargo delivery and insufficientunderstanding of the modes of their uptake. CPPs typically have an aminoacid composition that either contains a high relative abundance ofpositively charged amino acids such as lysine or arginine or hassequences that contain an alternating pattern of polar/charged aminoacids and non-polar, hydrophobic amino acids. These two types ofstructures are referred to as polycationic or amphipathic, respectively.A third class of CPPs are the hydrophobic peptides, containing onlyapolar residues, with low net charge or have hydrophobic amino acidgroups that are crucial for cellular uptake. The conjugation of the CPPto the peptide provided in the present invention can be performedfollowing well-known routine protocols, such as solid phase synthesis orsolution selective capping. (cf. Copolovici D. M. et al.,“Cell-Penetrating Peptides: Design, Synthesis, and Applications”, 2014,ACS Nano, 2014, 8 (3), pp 1972-1994).

In another embodiment of the second aspect of the invention, the peptideconsists of SEQ ID NO: 1.

In another embodiment of the second aspect of the invention, providedabove or below, the cell penetrating peptide is a polycationic CPP. Inanother embodiment of the second aspect of the invention, optionally incombination with any of the embodiments provided above or below, thecell penetrating peptide is polyArg.

In another embodiment of the second aspect of the invention, the peptideis of sequence SEQ ID NO: 15 (hereinafter also referred as “LH02”):

-   Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Val-Lys-Leu-Val-Asn-Leu-Gly-Phe-Ala-Thr-Leu-Arg-Glu-His-Val-Pro

In another embodiment, the peptide of the first or second aspect isconjugated to a label. In one embodiment, the label is conjugated to theN-terminal of the peptide.

A “label” as used herein is a molecule or compound that can be detectedby a variety of methods including fluorescence, electrical conductivity,radioactivity, size, and the like. The label may be intrinsicallycapable of emitting a signal, such as for example fluorescent label thatemits light of a particular wavelength following excitation by light ofanother lower, characteristic wavelength. Alternatively, the label maynot be capable of intrinsically emitting a signal but it may be capableof being bound by another compound that does emit a signal. An exampleof this latter situation is a label such as biotin which itself does notemit a signal but which when bound to labeled avidin or streptavidinmolecules can be detected. Other examples of this latter kind of labelare ligands that bind specifically to particular receptors. Detectablylabeled receptors are allowed to bind to ligand labeled unit specificmarkers in order to visualize such markers.

Labels that may be used according to the invention include but are notlimited to electron spin resonance molecule, a fluorescent molecule, achemiluminescent molecule, a radioisotope, an enzyme substrate, anenzyme, a biotin molecule, an avidin molecule, an electrical chargetransferring molecule, a semiconductor nanocrystal, a semiconductornanoparticle, a colloid gold nanocrystal, a ligand, a microbead, amagnetic bead, a paramagnetic molecule, a quantum dot, a chromogenicsubstrate, an affinity molecule, a protein, a peptide, nucleic acid, acarbohydrate, a hapten, an antigen, an antibody, an antibody fragment,and a lipid.

Radioisotopes can be detected with film or charge coupled devices(CCDs), ligands can be detected by binding of a receptor having afluorescent, chemiluminescent or enzyme tag, and microbeads can bedetected using electron or atomic force microscopy.

The conjugation of the label to the peptide can be performed followingroutine protocols well-known for the skilled in the art.

In another embodiment, the peptide of the first or second aspects isconjugated to a drug. In one embodiment, the drug is conjugated to theN-terminal end of the peptide.

Alternatively, the peptide of the invention can be conjugated to astabilising moiety, which enhances the biological properties of thepeptide, for example by improving its stability, solubility,bioavailibity and/or affinity. In another embodiment, optionally incombination with any of the embodiments provided above, the stabilisingmoiety is selected from mono or bifunctional poly(ethyleneglycol)(“PEG”), poly(vinyl alcohol) (“PVA”); other poly(alkylene oxides) suchas polypropylene glycol) (“PPG”); and poly(oxyethylated polyols) such aspoly(oxyethylated glycerol), poly(oxyethylated sorbitol), andpoly(oxyethylated glucose), and the like. In another embodiment of theinvention, optionally in combination with any of the embodimentsprovided above or below, the peptide of the invention is conjugated to apoly(ethyleneglycol). In another embodiment of the invention, optionallyin combination with any of the embodiments provided above or below, thepeptide of the invention is conjugated to a —(CH₂—O)₄— (hereinafter alsoreferred as “PEG4”).

In another embodiment, the peptide is of sequence SEQ ID NO: 16(hereinafter also referred as “H14-PEG4”):

The process for the preparation of the peptide according to the firstaspect of the invention comprises:

(1.a) the coupling, by condensation, of the corresponding amino acids ofthe peptide with a compound of formula (IV) and a compound of formula(V), which correspond to the amino acids referred as “i” and “i+4” or“i+7”. Compounds (IV) and (V) will be those suffering a subsequentcyclization step to generate the “L” biradical”:

wherein R₁₉ is as defined above, Z₁ and Z₂ are the same or different andrepresent (C₂-C₁₀)alkenyl; and

(1.b) a cyclization step comprising the ring-closed metathesis of Z₁ andZ₂ (cf. Kim Young-Woo et al., “Synthesis of all-hydrocarbon stapleda-helical peptides by ring-closing olefin metathesis”, Nature Protocols,2011, 6(6), p. 761-771; Scott J. M. et al., “Application of Ring-ClosingMetathesis to the Synthesis of Rigidified Amino Acids and Peptides”, J.Am. Chem. Soc., 1996, v. 118 (40), pp 9606-9614) performed in solutionwith a Grubbs (I or II generation) catalyst; or, alternatively,

(2a) the coupling, by condensation, of the required amino acids,including a compound of formula (VI) and a compound of formula (VII),which correspond to the amino acids referred as “i” and “i+4” or “i+7”.Compounds (VI) and (VII) will be those suffering a subsequentcyclization step to generate the “L” biradical”:

wherein R₁₉ is as defined above, Z₃ and Z₄ are the same or different,selected from the group consisting of: halogen —SH, —NHR₂₀, —OH,(C₂-C₁₀)alkyl-SH, (C₁-C₁₀)alkyl-OH, (C₁-C₁₀)alkyl-NHR₂₁, C(═O)OH,(C₁-C₁₀)C(═O)OH, C(═O)NHR₂₂, (C₁-C₁₀)alkylC(═O)NHR₂₃, OR₂₄,C(═O)-halogen, C(═O)—OR₂₅, S(═O)-halogen, S(═O)—OR₂₆, S(═O)₂R₂₇ whereR₂₀, R₂₁ R₂₂, R₂₃, R₂₄, R₂₅ R₂₆ and R₂₇ are monoradicals selected fromthe group consisting of: hydrogen, (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, and(C₂-C₁₀)alkinyl; a known ring system comprising from 3 to 14 carbonatoms, the system comprising from 1 to 3 rings, where:

-   -   each one of the rings is saturated, partially unsaturated, or        aromatic;    -   the rings are isolated, partially or totally fused,    -   each one of the members forming the known ring system is        selected from the group consisting of: —CH—, —CH₂—, —NH—, —N—,        —SH—, —S—, and —O—; and    -   the ring system is optionally substituted by one or more        radicals independently selected from the group consisting of        halogen, —OH, —NH₂, —SH, C(═O)-halogen (C₁-C₁₀)haloalkyl, and        (C₁-C₁₀)alkyl-O—; and

(2b) a cyclization step comprising the coupling reaction between Z₃ andZ₄ radicals; or, alternatively,

(3a) the coupling, by condensation, of the corresponding amino acids ofthe peptide with a compound of formula (VIII) and a compound of formula(IX), which correspond to the amino acids referred as “i” and “i+4” or“i+7”. Compounds (VIII) and (IX) will be those suffering a subsequentcyclization step to generate the “L” biradical”:

wherein R₁₉ is as defined above, one of Z₅ and Z₆ is (C₂-C₁₀)alkynyl andthe other is (C₂-C₁₀)alkylN₃; and

(3.b) a cyclization step comprising the condensation of Z₅ and Z₆radicals by well-known protocols such as the Cu(I)-mediated Huisgen1,3-dipolar cycloaddition reaction (a.k.a. a “click” reaction) togenerate a 1,4-substituted 1,2,3-triazole bridge (cf. Kolb H. C. et al.,“The growing impact of click chemistry on drug discovery”, 2003, DrugDiscov Today, 8(24):1128-1137).

The process for the preparation of the peptide according to the secondaspect of the invention comprises the coupling, by condensation, of thecarboxylic group or C-terminus of one amino acid with the amino group orN-terminus of another, this coupling reaction being repeated the numberof times required until the desired peptide is obtained. Alternatively,the peptide having the identity with sequence SEQ ID NO: 1 is preparedby coupling the required amino acids, and, in a second stage, thepeptide is fused to the cell penetrating peptide, following any of theprotocols known in the prior art (some of which have been previouslyreferred).

The compounds of formula (IV), (V), (VI), (VII), (VIII), and (IX) arecommercially available and are coupled by condensation to the alreadyformed portion of peptide sequence. These compounds can carry beads forthe appropriate solid phase synthesis of the peptide, as well asprotecting groups of the carboxy, amino or side-chain. Illustrativenon-limitative examples of compounds are: 2-(2′-propenyl)alanine,2-(3′-butenyl)glycine, 2-(4′-pentenyl)alanine, 2-(6′-heptenyl) alanine,2-(7′-octenyl)alanine, and allyl-glycine, 5-azido-norvaline,alpha-propargyl-alanine, among others.

The “coupling” step can be performed in solid phase, following theprotocol “deprotection-wash-coupling-wash”, by condensation of thecarboxylic group of one amino acid with the amino group of another aminoacid residue, using amino acids as defined above as well as alpha-alphadi-substituted amino acids of formula (IV) to (IX) in the order ofinterest to obtain the desired peptide.

The general principle of solid phase peptide synthesis is to repeatcycles of deprotection-wash-coupling-wash. The free N-terminal amine ofa solid-phase attached peptide is coupled to a single N-protected aminoacid unit. This unit is then deprotected, revealing a new N-terminalamine to which a further amino acid may be attached. Amino acids havereactive moieties at the N- and C-termini, which facilitates amino acidcoupling during synthesis. Many amino acids also have reactive sidechain functional groups, which can interact with free termini or otherside chain groups during synthesis and peptide elongation and negativelyinfluence yield and purity. To facilitate proper amino acid synthesiswith minimal side chain reactivity, chemical groups have been developedto bind to specific amino acid functional groups and block, or protect,the functional group from nonspecific reactions. These protectinggroups, while vast in nature, can be separated into three groups, asfollows: N-terminal protecting groups, C-terminal protecting groups(mostly used in liquid-phase synthesis), and side chain protectinggroups.

For coupling the peptides the carboxyl group is usually activated. Thisis important for speeding up the reaction. There are two main types ofactivating groups: carbodiimides and triazolols. However, the use ofpentafluorophenyl esters (FDPP, PFPOH]) and BOP—Cl are useful forcyclising peptides. Purified, individual amino acids are reacted withthese protecting groups prior to synthesis and then selectively removedduring specific steps of peptide synthesis.

Exemplary resins which may be employed by the present invention include,but are not limited to: (1) alkenyl resins (e.g., REM resin, vinylsulfone polymer-bound resin, vinyl-polystyrene resin); (2) aminefunctionalized resins (e.g., amidine resin,N-(4-Benzyloxybenzyl)hydroxylamine polymer bound,(aminomethyl)polystyrene, polymer bound (R)-(+)-a-methylbenzylamine,2-Chlorotrityl Knorr resin, 2-N-Fmoc-Amino-dibenzocyclohepta-1,4-diene,polymer-bound resin,4-[4-(1-Fmoc-aminoethyl)-2-methoxy-5-nitrophenoxy]butyramidomethyl-polystyreneresin, 4-Benzyloxybenzylamine, polymer-bound,4-Carboxybenzenesulfonamide, polymer-bound,Bis(tert-butoxycarbonyl)thiopseudourea, polymer-bound,Dimethylaminomethyl-polystyrene, Fmoc-3-amino-3-(2-nitrophenyl)propionicacid, polymer-bound, N-Methyl aminomethylated polystyrene, PAL resin,Sieber amide resin, tert-Butyl N-(2-mercaptoethyl)carbamate,polymer-bound, Triphenylchloromethane-4-carboxamide polymer bound); (3)benzhydrylamine (BHA) resins (e.g., 2-Chlorobenzhydryl chloride,polymer-bound, HMPB-benzhydrylamine polymer bound, 4-Methylbenzhydrol,polymer-bound, Benzhydryl chloride, polymer-bound, Benzhydrylaminepolymer-bound); (4) Br-functionalized resins (e.g., 4-(Benzyloxy)benzylbromide polymer bound, 4-Bromopolystyrene, Brominated PPOA resin,Brominated Wang resin, Bromoacetal, polymer-bound, Bromopolystyrene,HypoGel© 200 Br, Polystyrene A-Br for peptide synthesis, Seleniumbromide, polymer-bound, TentaGel HL-Br, TentaGel MB-Br, TentaGel S-Br,TentaGel S-Br); (5) Chloromethyl resins (e.g.,5-[4-(Chloromethyl)phenyl]pentyl]styrene, polymer-bound,4-(Benzyloxy)benzyl chloride polymer bound, 4-Methoxybenzhydrylchloride, polymer-bound); (6) CHO-functionalized resins (e.g.,(4-Formyl-3-methoxyphenoxymethyl)polystyrene,(4-Formyl-3-methoxyphenoxymethyl)polystyrene, 3-Benzyloxybenzaldehyde,polymer-bound, 4-Benzyloxy-2,6-dimethoxybenzaldehyde, polymer-bound,Formylpolystyrene, HypoGel© 200 CHO, Indole resin, PolystyreneA-CH(OEt)2, TentaGel HL-CH(OEt)2); (7) Cl-functionalized resins (e.g.,Benzoyl chloride polymer bound, (chloromethyl)polystyrene, Merrifield'sresin); N(8) CO2H functionalized resins (e.g., Carboxyethylpolystryrene,HypoGel® 200 COOH, Polystyrene AM-COOH, TentaGel HL-COOH, TentaGelMB—COOH, TentaGel S—COOH); (9) Hypo-Gel resins (e.g., HypoGel® 200 FMP,HypoGel® 200 PHB, HypoGel® 200 Trt-OH, HypoGel® 200 HMB); (10)I-functionalized resins (e.g., 4-lodophenol, polymer-bound,lodopolystyrene); Janda-Jes™ (JandaJel<ä>-Rink amide, JandaJel-NH₂,JandaJel-Cl, JandaJel-4-Mercaptophenol, JandaJel-OH,JandaJel-1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide,JandaJel-1,3,4,6,7,8-hexahydro-2H-pyrimido-[1,2-a]pyrimidine,JandaJel-morpholine, JandaJel-polypyridine, JandaJel-Triphenylphosphine,JandaJel-Wang); (11) MBHA resins (3[4′-(Hydroxymethyl)phenoxy]propionicacid-4-methylbenzhydrylamine resin, 4-(Hydroxymethyl)phenoxyacetic acidpolymer-bound to MBHA resin, HMBA-4-methylbenzhydrylamine polymer bound,4-Methylbenzhydrylamine hydrochloride polymer bound Capacity (amine));(12) NH2 functionalized resins ((Aminomethyl)polystyrene,(Aminomethyl)polystyrene, HypoGel® 200 NH2, Polystyrene AM-NH2,Polystyrene Microspheres 2-aminoethylated, Polystyrol Microspheres2-bromoethylated, Polystyrol Microspheres 2-hydroxyethylated, TentaGelHL-NH2, Tentagel M Br, Tentagel M NH2, Tentagel M OH, TentaGel MB—NH2,TentaGel S—NH2, TentaGel S—NH2); (13) OH-functionalized resins (e.g.,4-hydroxymethylbenzoic acid, polymer-bound, Hydroxymethyl Resins,OH-functionalized Wang Resins); (14) oxime resins (e.g.,4-Chlorobenzophenone oxime polymer bound, Benzophenone oxime polymerbound, 4-Methoxybenzophenone oxime polymer bound); (15) PEG resins(e.g., ethylene glycol polymer bound); (16) Boc-/Blz peptide synthesisresins (e.g., Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]-Cys(Acm)-b-Ala-O-PAM resin,Boc-Lys(Fmoc)-Lys[Boc-Lys(Fmoc)]-b-Ala-O-Pam resin,Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]-Lys{Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]}-b-Ala-O-PAMresin,Boc-Lys(Fmoc)-Lys[Boc-Lys(Fmoc)]-Lys[Boc-Lys(Fmoc)-Lys{Boc-Lys(Fmoc)]}-b-Ala-O-PAMresin,Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]-Lys{Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]}-Cys(Acm)-b-Ala-O-PAMresin, Preloaded PAM resins); (17) Fmoc-/t-Bu peptide synthesis resins(e.g., Fmoc-Lys(Fmoc)-Lys[Fmoc-Lys(Fmoc)]-b-Ala-O-Wang resin,Fmoc-Lys(Fmoc)-Lys[Fmoc-Lys(Fmoc)]-Lys{Fmoc-Lys(Fmoc)-Lys[Fmoc-Lys(Fmoc)]}-b-Ala-O-Wangresin, Preloaded TentaGel® S Trityl Resins, Preloaded TentaGel® Resins,Preloaded Trityl Resins, Preloaded Wang Resins, Trityl Resins Preloadedwith Amino Alcohols); (19) thiol-functionalized resins (e.g., HypoGel®200 S-Trt, Polystyrene AM-S-Trityl, TentaGel HL-S-Trityl, TentaGelMB—S-Trityl, TentaGel S—S-Trityl); and (20) Wang resins (e.g.,Fmoc-Ala-Wang resin, Fmoc-Arg(Pbf)-Wang resin, Fmoc-Arg(Pmc)-Wang resin,Fmoc-Asn(Trt)-Wang resin, Fmoc-Asp(OtBu)-Wang resin, Fmoc-Cys(Acm)-Wangresin, Fmoc-Cys(StBu)-Wang resin, Fmoc-Cys(Trt) Wang resin,Fmoc-Gln(Trt)-Wang resin, Fmoc-Glu(OtBu)-Wang resin, Fmoc-Gly-Wangresin, Fmoc-His(Trt)-Wang resin, Fmoc-Ile-Wang resin, Fmoc-Leu-Wangresin, Fmoc-Lys(Boc)-Wang resin, Fmoc-Met-Wang resin, Fmoc-D-Met-Wangresin, Fmoc-Phe-Wang resin, Fmoc-Pro-Wang resin, Fmoc-Ser(tBu)-Wangresin, Fmoc-Ser(Trt)-Wang resin, Fmoc-Thr(tBu)-Wang resin, Fmoc-Trp(Boc)Wang resin, Fmoc-Trp-Wang resin, Fmoc-Tyr(tBu)-Wang resin, Fmoc-Val-Wangresin).

“Protecting group” (PG) refers to a grouping of atoms that when attachedto a reactive group in a molecule masks, reduces or prevents thatreactivity.

Suitable amino-protecting groups include methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, Ntriphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Examples of suitably protected carboxylic acids further include, but arenot limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protectedcarboxylic acids. Examples of suitable silyl groups includetrimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples ofsuitable alkyl groups include methyl, benzyl, p-methoxybenzyl,3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl. Examples ofsuitable alkenyl groups include allyl. Examples of suitable aryl groupsinclude optionally substituted phenyl, biphenyl, or naphthyl. Examplesof suitable arylalkyl groups include optionally substituted benzyl(e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, 0-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2-and 4-picolyl.

In a third aspect, the present invention provides fusion peptidecomprising the peptide as defined in the first aspect of the inventionor any of the embodiments provided above.

In one embodiment of the third aspect of the invention, the fusionpeptide comprises the peptide as defined in the first aspect of theinvention, or in any of the embodiments of the first aspect of theinvention, and a cell penetrating peptide.

In another embodiment of the third aspect of the invention, the fusionpeptide is one of sequence SEQ ID NO: 17:

In a fourth aspect the present invention provides a pharmaceuticalcomposition.

The expression “therapeutically effective amount” as used herein, refersto the amount of a compound that, when administered, is sufficient toprevent development of, or alleviate to some extent, one or more of thesymptoms of the disease which is addressed. The particular dose of thepeptide administered according to this invention will of course bedetermined by the particular circumstances surrounding the case,including the compound administered, the route of administration, theparticular condition being treated, and the similar considerations.

The expression “pharmaceutically acceptable excipients or carriers”refers to pharmaceutically acceptable materials, compositions orvehicles. Each component must be pharmaceutically acceptable in thesense of being compatible with the other ingredients of thepharmaceutical composition. It must also be suitable for use in contactwith the tissue or organ of humans and non-human animals withoutexcessive toxicity, irritation, allergic response, immunogenicity orother problems or complications commensurate with a reasonablebenefit/risk ratio. Examples of suitable pharmaceutically acceptableexcipients are solvents, dispersion media, diluents, or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like. Except insofar as any conventional excipientmedium is incompatible with a substance or its derivatives, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in bulk, as a single unit dose, and/or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient (peptide as defined in anyof the previous aspects and embodiments), the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in the inventive formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, andcombinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpolyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,and combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminumsilicate]), long chain amino acid derivatives, high molecular weightalcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.,carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters {e.g., polyoxyethylenesorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60],polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate[Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span65], glyceryl monooleate, sorbitan monooleate [Span 80]),polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., Cremophor), polyoxyethyleneethers, (e.g., polyoxyethylene lauryl ether [Brij 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.,cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural andsynthetic gums (e.g., acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,polyvinylpyrrolidone), magnesium aluminum silicate (Veegum), and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; and combinations thereof.

Exemplary preservatives may include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives. Exemplaryantioxidants include, but are not limited to, alpha tocopherol, ascorbicacid, acorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, monothioglycerol, potassium metabisulfite, propionicacid, propyl gallate, sodium ascorbate, sodium bisulfite, sodiummetabisulfite, and sodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservativesinclude, but are not limited to, vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid. Other preservatives include, but arenot limited to, tocopherol, tocopherol acetate, deteroxime mesylate,cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ethersulfate (SLES), sodium bisulfite, sodium metabisulfite, potassiumsulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben,Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certainembodiments, the preservative is an anti-oxidant. In other embodiments,the preservative is a chelating agent.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, and combinationsthereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable liposomes emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredients, the liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions caninclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents. In certainembodiments for parenteral administration, the conjugates of theinvention are mixed with solubilizing agents such as polyethoxylatedcastor oil (e.g. CREMOPHOR™), alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.Alternatively, the preparation can be in the form of liposomes.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The peptides of the invention can be in micro-encapsulated form with oneor more excipients as noted above. In one embodiment, the peptides ofthe invention are formulated in liposomes. The solid dosage forms oftablets, dragees, capsules, pills, and granules can be prepared withcoatings and shells such as enteric coatings, release controllingcoatings and other coatings well known in the pharmaceutical formulatingart. In such solid dosage forms the active ingredient may be admixedwith at least one inert diluent such as sucrose, lactose or starch. Suchdosage forms may comprise, as is normal practice, additional substancesother than inert diluents, e.g., tableting lubricants and othertableting aids such a magnesium stearate and microcrystalline cellulose.In the case of capsules, tablets and pills, the dosage forms maycomprise buffering agents. They may optionally comprise opacifyingagents and can be of a composition that they release the activeingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions which can be used include polymeric substances and waxes.

It will be appreciated that peptides and pharmaceutical compositions ofthe present invention can be employed in combination therapies. Theparticular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will be appreciated that the therapiesemployed may achieve a desired effect for the same purpose (for example,an inventive conjugate useful for detecting tumors may be administeredconcurrently with another agent useful for detecting tumors), or theymay achieve different effects (e.g., control of any adverse effects).

The pharmaceutical composition of the present invention may beadministered either alone or in combination with one or more othertherapeutic agents. By “in combination with,” it is not intended toimply that the agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope of the invention. The compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In general, each agent will beadministered at a dose and/or on a time schedule determined for thatagent. Additionally, the invention encompasses the delivery of thepeptide or pharmaceutical compositions in combination with agents thatmay improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

The particular combination of therapies to employ in a combinationregimen will take into account compatibility of the desired therapeuticsand/or procedures and/or the desired therapeutic effect to be achieved.It will be appreciated that the therapies employed may achieve a desiredeffect for the same disorder (for example, an inventive polypeptide maybe administered concurrently with another biologically active agent usedto treat the same disorder), and/or they may achieve different effects(e.g., control of any adverse effects). In will further be appreciatedthat biologically active agents utilized in this combination may beadministered together in a single composition or administered separatelyin different compositions.

The expression “in combination with” also encompasses the possibility ofconjugating (by chemical-physical interactions) the peptide of theinvention to any of the further agents mentioned above and below, whichcan be either a therapeutic agent or an agent for improving the profileof the peptide (such as bioavailability), among others.

In one embodiment, the peptides of the invention are administered incombination with one or more anti-cancer agents. An anti-cancer agentmay be, for instance, methotrexate, vincristine, adriamycin, cisplatin,non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycinC, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA,valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RASfarnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP,MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone,Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340,AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX895 if,Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin,Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomaldoxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609(754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer,Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed,Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin,Caelyx/liposomal doxorubicin, Fludara/Fludarabine,Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD0473/Anormed, YM 116, iodine seeds, CDK4 and CDK2 inhibitors, PARPinhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog,nitrosoureas, alkylating agents such as melphelan and cyclophosphamide,Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil,Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphatesodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU),Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, InterferonAlfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue),Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),Mercaptopurine, Mesna, Mitotane (o.p-DDD), Mitoxantrone HCl, Octreotide,Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate,Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA),Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2,Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG),Pentostatin (2′-deoxycoformycin), Semustine (methyl-CCNU), Teniposide(VM-26) or Vindesine sulfate, signal transduction inhibitors (such asMEK, BRAF, AKT, her2, mTOR, and PI3K inhibitors), but it is not solimited.

As it is illustrated below, the peptides of the invention are useful inthe treatment of a cancer selected from the group consisting of:leukemia, breast cancer, glioblastoma, and lung cancer.

Throughout the description and claims the word “comprise” and variationsof the word, are not intended to exclude other technical features,additives, components, or steps. Furthermore, the word “comprise”encompasses the case of “consisting of”. Additional objects, advantagesand features of the invention will become apparent to those skilled inthe art upon examination of the description or may be learned bypractice of the invention. The following examples are provided by way ofillustration, and they are not intended to be limiting of the presentinvention. Furthermore, the present invention covers all possiblecombinations of particular and preferred embodiments described herein.

EXAMPLES

Synthetic General Procedure

Compounds LH1 and LH2

Materials were purchased as following: Fmoc-protected α-amino acids;Rinkamide MBHA Resin (Tianjin Nankai HECHENG S&T Co., Ltd); HBTU((2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), GL Biochem); N-methyl morpholine (SinopharmChemical Reagent Co., Ltd.); Succinic anhydride (Aladdin); acetonitrile(Xingke Chemical); ninhydrin (Sinopharm Chemical Reagent Co., Ltd.);Piperidine (Vertellus); Dimethylformamide, DMF (Zhejiang jiangshanchemical co., Ltd); trifluoroacetic acid, TFA (Trifluoroacetic acid,Solvay), TIS (Thioanisole, Solvay)

Briefly, the linear polypeptides were synthesized manually using Fmocbased SPPS (solid phase peptides synthesis) on Rink amide MBHA resin assupport.

The following protocol was used:

-   -   1. The Fmoc protective group was removed with 20% piperidine in        DMF.    -   2. The resin was washed with DMF five times.    -   3. The subsequent Fmoc-protected amino acid was coupled for 45        min using Fmoc-AA (3 equiv.), HBTU (3 equiv.), and N-methyl        morpholine (6 equiv.).    -   4. The resin was washed with DMF five times. Coupling was        checked by ninhydrin test.    -   5. Repeat from step 1.    -   6. N-terminal was capped by reacting with succinic anhydride (10        equiv.) and N-methyl morpholine (10 equiv.).

The peptide was cleaved from the resin and deprotected by exposure tosolution F (95% TFA, 2,5% water, 2.5% TIS) and lyophilized.

The lyophilized peptides were purified by reverse phase HPLC using a C18column (see compounds characterization for details). The peptides wereidentified by LC-MS-ESI. All the mass spectral data for all thecompounds are shown below in Table 1.

Compounds H02, H04, H14, H14-A09, H14-A12, H14-F12, H14-F14, H14-h2-01,H14-Q06, H14-Q09, H14-Q13, H14-Q15, H14-S15, H14-CPP1 and H14-PEG4.

Materials were purchased as following: Fmoc-protected α-amino acids, theFmoc-protected olefinic amino acids: Fmoc-[(S)-2-(4 pentenyl)alanine]OH,Fmoc-[(R)-2-(4 pentenyl)alanine]OH, Fmoc-[(S)-2-(7 octenyl)alanine]OH,Fmoc-[(R)-2-(4 pentenyl)alanine]OH, FMoc-PEG4-COOH and Boc-PEG4-COOH,2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (TBTU), resins, dimethylformamide (DMF),N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA),1,2-dichloroethane (DCE), Grubbs Ru(IV) catalyst and piperidine werepurchased from different suppliers.

Briefly, the linear polypeptides were synthesized with automaticsynthesizer using Fmoc solid phase peptide chemistry. The N terminalFMoc-PEG4 or Boc-PEG4 were added to the sequence as a regularaminoacids. Only the coupling with olefinic amino acids was performedmanually after removing the resins from the reactor vessel, as disclosedin the previous section.

The ring-closing metathesis reaction was performed in solution with afirst generation Grubbs catalyst after cleaving the linear peptide fromthe resin, as disclosed by Scott J. M. and colleagues (Scott J. M. etal., “Application of Ring-Closing Metathesis to the Synthesis ofRigidified Amino Acids and Peptides”, 1996, J. Am. Chem. Soc., 1996, 118(40), pp 9606-9614).

The deprotected peptide precipitated with methyl-tert-butyl ether at 4°C. and lyophilized.

The lyophilized peptides were purified by reverse phase HPLC using a C18column (see compounds characterization for details). The peptides wereidentified by LC-MS-ESI. All the mass spectral data for all thecompounds are shown below in Table 1.

HPLC conditions:

LH01. The compound was purified by HPLC-RP (C-18 column; Pump A: H₂Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 29%-39% of B in 20 minutes (R. T.=9.97). Purity grade 95.70% byHPLC;

LH02. The compound was purified by HPLC-RP (C-18 column; Pump A: H₂Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 27%-37% of B in 20 minutes (R. T.=10.92). Purity grade 98.17%by HPLC;

H02. The compound was purified by HPLC-RP (C-18 column; Pump A: H₂O with0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a linear gradient5%-60% of B in 11 minutes (R. T.=6.72). Purity grade 96.98% by HPLC;

H04. The compound was purified by HPLC-RP (C-18 column; Pump A: H₂O with0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a linear gradient5%-60% of B in 15 minutes (R. T.=7.47). Purity grade 90.90% by HPLC;

H14. The compound was purified by HPLC-RP (C-18 column; Pump A: H₂O with0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a linear gradient10%-100% of B in 8 minutes (R. T.=5.97). Purity grade 95.00% by HPLC.

H14-A09: The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 52%-62% of B in 20 minutes (R. T.=7.26-9.15). Purity grade95.07% by HPLC;

H14-A12. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 60%-70% of B in 20 minutes (R. T.=8.80). Purity grade 91.04% byHPLC;

H14-F12. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 58%-78% of B in 20 minutes (R. T.=12.33). Purity grade 96.84%by HPLC;

H14-F14. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 62%-72% of B in 20 minutes (R. T.=13.82-15.43). Purity grade98.43% by HPLC;

H14-h2-01. The compound was purified by HPLC-RP (C-18 column; Pump A:H2O with 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 56%-76% of B in 20 minutes (R. T.=9.49 y 11.69). Purity grade96.40% by HPLC;

H14-Q6. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 42%-62% of B in 20 minutes (R. T.=14.75). Purity grade 90.00%by HPLC;

H14-Q09. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 60%-80% of B in 20 minutes (R. T.=6.77-7.99). Purity grade95.35% by HPLC;

H14-Q13. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 48%-68% of B in 20 minutes (R. T.=7.2-9.14). Purity grade95.00% by HPLC;

H14-Q15. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 58%-78% of B in 20 minutes (R. T.=9.86). Purity grade 95.00% byHPLC; and

H14-S15. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 65%-85% of B in 20 minutes (R. T.=6.89). Purity grade 95.97% byHPLC.

H14-CPP1. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 48%-68% of B in 20 minutes (R. T.=12.35). Purity grade 95.04%by HPLC;

H14-PEG4. The compound was purified by HPLC-RP (C-18 column; Pump A: H2Owith 0.1% TFA; Pump B Acetonitrile with 0.1% TFA) using a lineargradient 58%-78% of B in 20 minutes (R. T.=9.44-10.91). Purity grade93.07% by HPLC;

TABLE 1 mass characterization SEQ ID MW Mass Mass Mass Mass NO: N. IDSequence (1H) (2H) (3H) (4H) (5H) SEQ ID IDP- RVKLVNLGFATLRE calcul.1948.3 975.2 NO: 1 LH01 HVP found 972.8 SEQ ID IDP- RRRRRRRRVKLVNcalcul. 3041.7 1014.9 761.4 609.3 NO: 15 LH02 LGFATLREHVP found 1015.0761.5 609.4 SEQ ID IDP- RNRVKXVNLXFATL calcul. 2102.3 1052.2 701.8 NO: 2H02 REH found 1052.4 702.1 SEQ ID IDP- RNRVKLVNLXFATL calcul. 2120.31061.2 707.8 NO: 3 H04 REX found 1061.5 708.0 SEQ ID IDP- RVKLVNLXFATLREcalcul. 2046.5 1024.3 683.2 NO: 4 H14 XVP found 1023.8 683.1 SEQ ID NO:IDP-H14-A09 RVKLVNLXFAALR calcul. 1988.5 995.3 663.8 5 EXVP found 995.2663.9 SEQ ID NO: IDP-H14-A12 RVKLVNLXFATLR calcul. 1988.5 995.3 663.8 6AXVP found 995.2 663.9 SEQ ID NO: IDP-H14-F12 RVKLVNLXFATLR calcul.2064.6 1033.3 689.2 7 FXVP found 1033.2 689.2 SEQ ID NO: IDP-H14-F14RVKLVNLXFATLR calcul. 2096.6 1047.3 8 EXVF found 1046.4 SEQ ID NO:IDP-H14-h2- RVKLVNLXFQALR calcul. 2072.6 1037.3 691.9 9 01 QXVP found1037.3 691.9 SEQ ID NO: IDP-H14-Q06 RVKLVQLXFATLR calcul. 2061.2 1031.6688.1 10 EXVP found 1031.3 687.9 SEQ ID NO: IDP-H14-Q09 RVKLVNLXFQTLRcalcul. 1988.5 995.3 663.8 11 EXVP found 995.2 663.9 SEQ ID NO:IDP-H14-Q13 RVKLVNLXFATLR calcul. 2046.2 1024.1 683.1 12 QXVP found1023.8 682.9 SEQ ID NO: IDP-H14-Q15 RVKLVNLXFATLR calcul. 2078.2 1040.1693.7 13 EXVQ found 1039.8 693.6 SEQ ID NO: IDP-H14-S15 RVKLVNLXFATLRcalcul. 2037.1 1017.5 14 EXVS found 1016.8 SEQ ID NO: IDP-H14-PegRVKLVNLXFAT calcul. 2294.7 765.9 16 PEG4 LREXVP found 765.6 SEQ IDNO: IDP-14-CPP1 R7- calcul. 3139.8 1570.9 1047.6 17 RVKLVNLXFATLR found1570.9 1047.4 EXVP

The X biradical means:

and the L biradical means —(CH₂)₃—CH═CH—(CH₂)₃— (for peptide H02) or—(CH₂)₆—CH═CH—(CH₂)₃— (for peptides H04, H14, H14-A12, H14-F12, H14-F14,H14-h2-01, H14-Q06, H14-Q09, H14-Q13, H14-Q15, and H14-S15).

Cell Lines:

H5B, epithelial small cell lung cancer extracted from SCLC tumor of GEMM(p53−/− y Rb−/−) mice (provided by Stanford University);

HL-60, promyeloblast (acute myelocytic leukemia, AML), ECACC: 98070106;

MCF-7, epithelial (breast cancer), ECACC: 86012803;

GMB-18, epithelial (glioblastoma, GMB), from surgical specimens frompatients;

GMB-27, epithelial (glioblastoma, GMB), from surgical specimens frompatients;

NCI-H524, lung (small cell lung cancer, SCLC), ATCC-CRL-5831;

NCI-H526, epithelial (small cell lung cancer, SCLC), ATCC-CRL-5811;

NCI-69, (small cell lung cancer, SCLC), ATCC-HTB-119;

NCI-H82, epithelial (small cell lung cancer, SCLC), ATCC-CRL-5811;

NCI-H187, epithelial (small cell lung cancer, SCLC), ATCC-CRL-5804;

NCI-510A, epithelial (small cell lung cancer, SCLC), ATCC-HBT-124; andHepatocytes (normal liver), from a woman donor was provided byFoundation DTI-Donation & Transplantation Institute from surgical wastesand from organs not suitable for transplantation

Cell Culture

Cell line MCF-7 was cultured in incubator under CO₂ (6%) at 37° C. inDMEM high glucose (Dulbecco's Modified Eagle Solution, Gibco-BRL31966-21) medium with 10% fetal bovine serum inactivated (FBS)(Gibco-BRL 10106-169). Cell lines HL-60, NCI-H524, NCI-H526, NCI-H69,NCI-H82, NCI-H187 and H5B were cultured in an incubator at 37° C. inRPMI-1640 (Sigma R8758) medium with 10% of fetal bovine seruminactivated (FBS) and 2 mM glutamine (Sigma G7513). Cell line NCI-H510Awas cultured in incubator in F12K medium with 10% of fetal bovine seruminactivated (FBS). Cell line GBM-18 and GBM-27 were cultured inincubator under CO₂ (6%) at 37° C. in complete medium(Neurobasal+B27+Glutamax+Growth factors).

During the amplification step and the assays, adherent cells were rinsedwith DPBS (Dulbecco's Phosphate Buffered Saline, Sigma D1283) threetimes and afterward treated for 5 minutes with trypsin ([0.5 g/ml]/EDTA[0.2 g/ml]) (Gibco-BRL, 15400054) in solution of DPBS at 37° C., and,once detached, transferred in the culturing medium. No-adherent cellswere centrifuged and transferred in the culturing medium. Cells werecounted in a Neubauer chamber after labelling with Tripan-Blue. Eachassay was performed only when the viability was superior to 90%.

Human liver from a woman donor was subjected to hepatocyte isolation byusing a two-step tissue microperfusion technique taking maximum care toavoid anoxia before cannulation, since hepatocytes are cells highlysensitive to oxygen levels. An initial anoxic state could result in alow viability of the cells, and will later affect their metaboliccapacity in the culture. Briefly, liver tissue was cannulated by usingthe accessible vessels with a 1 mm-diameter needle. A balanced saltsolution was injected to clean the organ by using a peristaltic pumpadjusted to a flow rate of 18-20 ml/min. After washing, a solutioncontaining collagenase was added for liver disintegration. The cellsuspension obtained using this process was filtered and centrifuged and,after a couple of washings to remove collagenase, cells werere-suspended in culture medium Ham's F-12/William (1:1) mediumsupplemented with 2 mM glutamine, 170 μg/ml sodium selenite, 2% newborncalf serum, 50 mU/ml penicillin, 50 μg/ml streptomycin, 0.1% bovineserum albumin, 1.68 mM NAME, 26 mM sodium bicarbonate, 25 μg/mltransferrin, 65.5 μM ethanolamine, 7.2 μM linoleic acid, 17.5 mMglucose, 6.14 mM ascorbic acid and 10 nM insulin.

After resuspension, a cell aliquot was removed and viability wasdetermined by cell counting using the trypan blue exclusion method.Trypan blue uptake is the result of cell membrane alteration and inconsequence of cell death. Thus, to quantify cell viability, 0.4% trypanblue in saline was added to the cells and these were immediately loadedin a Neubauer chamber. Viable cells were counted in 5 different fieldsunder the optical microscope as non-blue cells.% Viability=Number of non-blue cells×100/Number of total cellsViability and Hepatoxicity Assay

MCF-7, NCI-H187, NCI-H69, NCI-H526 and H5B cells were seeded at adensity of 5000 cells/well; NCI-H524 and NCI-H82 were seeded at adensity of 2500 cells/well; HL-60 and NCI-H510A at 10000 cells/well in100 μl of medium in 96 well plates. After 24 h, the compounds to betested were added to calculate the dose/response curve at the startingconcentration of 100 μM with serial dilutions (1:1). Controls were theuntreated cells. Each experiment was performed in triplicate.

Cells were incubated during 72 h in incubator under CO₂ atmosphere at37° C. Cell viability was measured by means of MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay,Alamar Blue® (Biosource DAL1100) and Hexosaminidase activity test,consecutively, following manufacturer instructions.

Assay Proceeding Follows:

MTT: 1. Stock solution of MTT (475989 Calbiochem) was 5 mg/ml in PBS 1×.As negative control (experiment noise) 3 wells were treated with 20μl/well of a solution of SDS 10% in H₂O. The same control was used forAlamar Blue/Hexosaminidase. 10 μl/well of MTT solution were added andthe plate was incubated for 3-4 hr. The medium was discarded and 100 μlof extracting buffer (PBS 1×, 15% SDS, 50% Na N,N-Dimethylformamide, pH4,7) were added to each well. Plates were incubated for 16 h at roomtemperature under orbital shaking. Absorbance at 570 nm was finallymeasured.

Alamar Blue: 10 μl Alamar Blue solution was added to each well and theplates were incubated for 4 in the incubator. Fluorescence ratio at535/590 (excitation/emission) was measured in Cytofluor (Millipore)fluorimeter. Blank control was determined by lysis of untreated cellswith con 2% of Triton X100, right before adding Alamar Blue solution.

Hexosaminidase activity test: after Alamar Blue lecture, medium wasdiscarded and plates were rinsed once with PBS. 60 μl hexosaminidasesubstrate (p-nitrophenol-N-acetyl-beta-D-glucosamide 7.5 mM [SigmaN-9376], sodium citrate 0.1 M, pH 5.0, 0.25% Triton X-100) was added toeach well and the plates were incubated at 37° C. for 2-5 h, accordingto the cell type (hexosaminidase activity changes according to the celltype). After incubation time, 90 μl of revealing solution (Glycine 50mM, pH 10.4; EDTA 5 mM) were added to each well, and absorbance at 410nM was measured. Blank control was the same as described before.

Human hepatocytes isolated and re-suspended as described before wereseeded at 100,000 viable cells/well on 96 collagen-coated plates inorder to assess compound's cytotoxicity. After 1 hour in culture, mediumwas renewed to eliminate cell debris. Cultured human hepatocytes wereallowed to stay in culture for 24 hours prior performing theexperiments. After 24 h, the compounds to be tested were added tocalculate the dose/response curve at the starting concentration of 400μM with serial dilutions. Each experiment was performed in triplicate.Cells were incubated during 24 h in incubator under CO₂ atmosphere at37° C.

The viability was determined by cell counting using the trypan blueexclusion method.% Viability=Number of non-blue cells×100/Number of total cellsStatistics

Data analysis was performed calculating the percentage of cell viabilitynormalized vs. the values of negative control, which was consideredequal to 100%. The dose/response curve was fitted through the sigmoidalequation dose-response (variable slope) and the EC₅₀ values werecalculated as follow:Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}{[(LogEC50−X)*HillSlope]},

where: X is compound concentration (log scale) and Y is the response

Calculations and graphs were conducted using GraphPad Prism (Prism 6 forWindows).

Mechanism of Action—Western Blot Activated Caspase 3, Parp and c-Parp

Cells were incubated during 6 h and 12 h in incubator under CO₂atmosphere at 37° C. with a range of concentration of H14 (1 μM and 10μM).

Samples containing equal amounts of proteins extracted from lysates ofcultured cell lines NCI-H187 and NCI-H510A (5-50 ug depending on thespecification of the corresponding primary antibody, Santa CruzBiotechnologies, Santa Cruz, Calif.) underwent electrophoresis onSDS-PAGE and were transferred to polyvinylidene difluoride membranes.The membranes were blocked in 3% BSA-PBS-0.1% Tween at room temperaturefor 1 h and blots were probed over night at 4° C. with primaryantibodies (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to detectproteins of interests. After incubation, the membranes were washed threetimes with wreashing buffer (PBS containing 0.1% Tween) for 5 min.Membranes were then incubated for 1 h with 1:10,000 diluted secondaryantibodies (Santa Cruz Biotechnologies, Santa Cruz, Calif.) at roomtemperature. Specific proteins were detected using G-Box (Syngene,Cambridge, UK) after washing.

Stability Test

Chemicals

-   -   Water, Purelab Ultra Ionic (Elga Berkefeld GmbH, Celle, Germany)    -   Methanol, for HPLC, ≥99.9% (Sigma Aldrich GmbH, Buchs,        Switzerland)    -   Acetonitrile, for HPLC, ≥99.9% (Sigma Aldrich GmbH, Buchs,        Switzerland)    -   2 Propanol, for HPLC, ≥99.9% (Sigma Aldrich GmbH, Buchs,        Switzerland)    -   Ethanol, for HPLC, ≥99.8% (Sigma Aldrich GmbH, Buchs,        Switzerland)    -   Formic acid, puriss. p.a., ≥98% (Sigma Aldrich GmbH, Buchs,        Switzerland)        Blank Matrix    -   Human plasma with K3 EDTA as anticoagulant (Blutspendezentrum        SRK beider Basel, Basel, Switzerland)        Apparatus    -   Centrifuge, Allegra 64R (Beckman Coulter International SA, Nyon,        Switzerland)    -   Centrifuge, Biofuge Stratos (Thermo Fisher Scientific, Osterode,        Germany)    -   Freezer, Liebherr Comfort (Liebherr, Biberach an der Riss,        Germany)    -   Deep freezer, MDF U53V (SANYO Electric Co. Ltd., Tokyo, Japan)    -   Ultrasonic bath, SW 6 H (Sonoswiss AG, Ramsen, Switzerland)    -   Pipette, Reference, Research (Eppendorf AG, Hamburg, Germany)    -   Vortexer, Genius 3 (IKA Labortechnik, Staufen, Germany)    -   Vortexer, Genie 2 (Scientific Industries Inc., New York, USA)    -   Micro balance, MT 5 (Mettler Toledo GmbH, Giessen, Germany)    -   Precision balance, PB503 S/FACT (Mettler Toledo GmbH, Giessen,        Germany)    -   Thermomixer comfort (Eppendorf AG, Hamburg, Germany)        Instrumentation    -   HPLC pump, 1200 series pump (Agilent Technologies Inc, Santa        Clara, Calif., USA)    -   Autosampler, HTC xt PAL (CTC Analytics AG, Zwingen, Switzerland)    -   Mass spectrometer, TSQ Vantage (Thermo Fisher Scientific, San        Jose, Calif., USA)        Analytical Method        a. Preparation of Calibration Samples and Quality Control        Samples

For the preparation of stock solutions, H14 was dissolved inwater/acetonitrile (95/5, v/v) containing 0.1% formic acid to aconcentration of 1.00 mg/mL. Further serial dilutions were carried outin plasma. The concentrations were calculated under consideration ofpurity.

For preparation of plasma samples, the human blank plasma were fortifiedwith the stock solution using a ratio of 1:100 to obtain a sample with10.0 μg/mL which was subsequently serial-diluted in human blank plasma.

The concentrations of the samples in plasma were the following:

TABLE 2 Human Sample 1 Human Sample 2 Analyte [ng/mL] [ng/mL] H14 1001000b. Preparation of the Internal Standard Solution

For the preparation of internal standard stock solution, the internalstandard was dissolved in water/acetonitrile (95/5, v/v) containing 0.1%formic acid to a concentration of 1.00 mg/mL. Final working dilutionswas prepared to a concentration of 1000 ng/mL in acetonitrile containing1% formic acid for the analysis of plasma samples.

c. Sample Preparation

A volume of 300 μL of human plasma samples for the two peptideconcentrations (100 ng/mL and 1000 ng/mL) were transferred to an empty96 deep well plate. The plate was incubated at 37° C. in a thermomixerat approximately 700 rpm. At selected incubation time points of 0 h, 1h, 2 h, 4 h, 6 h and 24 h, aliquots of plasma were precipitated withacetonitrile containing internal standard. To an aliquot of 20 μL humanplasma samples, 40 μL of acetonitrile containing 1% formic acid and theinternal standard was added. After vortex mixing for a few seconds, thesamples were centrifuged for about 10 minutes at approximately 50000 g.The temperature of the centrifuge was set to 8° C. An aliquot of 30 μLof the supernatant was transferred to an autosampler vial for analysisusing LC MS/MS.

c. HPLC MS/MS Conditions

The quantification of H14 was performed by column separation withreverse phase chromatography followed by detection with triple stagequadrupole MS/MS in the selected reaction monitoring mode. For HPLC thecondition were the following:

Injection volume 3 μL (5 μL into a loop of 3 μL)Mobile Phase:

Phase A water containing 0.5% formic acid Phase B 2 propanol containing0.5% formic acidColumn:

-   -   Hypersil Gold, 2.1×50 mm, 3 μm (Thermo Fisher Scientific Inc.,        Waltham, Mass., USA)

Column temperature: room temperature

Gradient:

TABLE 3 Time Flow Phase A Phase B [min] [mL/min] [%] [%] 0.00 0.400 98.02.00 0.10 0.400 98.0 2.00 2.40 0.400 5.00 95.0 4.00 0.400 5.00 95.0 4.100.400 98.0 2.00 5.00 0.400 98.0 2.00

Mass spectrometry Ion source HESI Polarity Positive Voltage [V] 3500Sweep gas [au] 0.0 Sheath gas [au] 60.0 Auxiliary gas [au] 5.0 Vaporizer[° C.] 300 Collision gas pressure [mTorr] 1.5Selected Reaction Monitoring:

TABLE 4 parent ion product ion width Scan time collision analyte mass[mu] mass [mu] [mu] [s] energy [V] H14 683.200 602.600 0.100 0.050 18In Vivo Studies

Animals:

Foxn1nu immunosuppressed mice of 6-7 weeks old housed and handled in apathogen-free zone (CIMA University of Navarra). All experiments werecarried out within the facilities of the University of Navarra.

Groups studied:

-   -   Control: Medium (PBS) i.p. each 24 hours each day.    -   IDP-H14 20 mg/kg i.p. each 24 hours from each day.

Methods and follow-up of the study:

Subcutaneous xenograft model: The mice were shaved on the right and leftflanks and anesthetized by inhalation to decrease their mobility, wereinoculated subcutaneously with H510A cells in 50 μl of RPMI-1640 mediumand 50 μl of Corning® Matrigel® Basement Membrane Matrix. When thetumors became palpable, the mice were randomized into the differentgroups (8 mice in the control group and 8 in treated group) according tothe tumor volume. This was estimated by measurements with a caliper ofthe two tumor diameters and using the following formula of a spheroid:V=(a·b{circumflex over ( )}2·π)/6

where a and b correspond to the longest and shortest diameter,respectively. Tumor volume was monitored three times per week.

Survival evaluation: Mice were sacrificed when their tumor diameterreached a volume of 1000 mm³. The time to reach the endpoint criterionwas estimated from the day of treatment start. The statisticaldifferences were evaluated using Kaplan-Meier curves with the log ranktest. Statistical analyzes were performed using the SPSS-17.0 program.

2. Results

2.1 Efficacy Results

The peptides of the invention were found to be highly efficient ininhibiting the proliferation of different lung cancer cells. Theexperimental data are summarized in Table 5 below:

TABLE 5 EC₅₀ (μM) Cell line LH01 LH02 H02 H04 H14 H-82 >100 ≈7 n.t. n.t.≈12.5 H-187 >100 ≈10 n.t. n.t. ≈10 H-524 n.t. n.t. ≈15  ≈9 ≈12.5 H-526n.t. n.t. ≈10 ≈11 n.t. H-69 n.t. n.t. n.t. n.t. ≈7 H-510A n.t. n.t. n.t.n.t. ≈12 LH01 is used for comparative purposes; n.t. = not-tested

Peptide of the invention H14 was also tested to confirm itsantiproliferative action in other cancer cell lines, such as breast,leukemia cell and glioblastoma lines. Surprisingly, it was found thatthe peptide was active, as provided in Table 6 below:

TABLE 6 EC₅₀ (μM) MCF-7 HL-60 GBM-18 GMB-27 H14 ≈9 ≈14 ≈4 ≈5

In addition, H14 mutants peptide (i.e. peptides H14-09, H14-A12,H14-F12, H14-F14, H14-h2-01, H14-Q06, H14-Q09, H14-Q13, H14-Q15, andH14-S15) were also found to be active against several cancer cell lines,as it is shown in Tables 7 and 8:

TABLE 7 Active compound (EC₅₀) Cell IDP-H14- IDP-H14- IDP-H14- IDP-H14-IDP-H14- line A09 A12 F12 F14 H2-01 H-5B nt ≈2.5 ≈2.5 ≈7.5 ≈2.5 H-82≈7.5 ≈7.5 ≈7.5 ≈7.5 ≈7.5 H-187 ≈7.5 ≈7.5 ≈7.5 ≈7.5 ≈7.5 H-510A ≈7.5 ≈7.5≈7.5 ≈7.5 ≈7.5 EC₅₀ expressed in μM

TABLE 8 Active compound (EC₅₀) Cell IDP-H14- IDP-H14- IDP-H14- IDP-H14-IDP-H14- line Q6 Q9 Q13 Q15 S15 H-5B ≈7.5 nt ≈7.5 ≈7.5 ≈2.5 H-82 ≈7.5 nt≈7.5 ≈7.5 ≈7.5 H-187 ≈7.5 ≈12.5 ≈7.5 ≈7.5 ≈7.5 H-510A ≈12.5 nt ≈12.5≈7.5 ≈7.5 EC₅₀ expressed in μM

Surprisingly, with the punctual mutations the resulting peptides showedan improved efficacy vs H14.

When peptide H14 was conjugated to a CPP, the efficacy was alsosubstantially improved, as it is shown in Table 9:

TABLE 9 Active compound (EC₅₀) Cell line IDP-H14-CPP H-5B ≈7.5 H-82 ≈7.5H-187 ≈7.5 H-510A ≈7.5 EC₅₀ expressed in μM2.2 Hepatotoxicity

The experimental data are summarized in Table 10 below:

TABLE 10 H14 (μM) % Viability SD Untreated 100.0 4.5 0.5% SDS 13.6 0.1 4 112.9 10.0 14 119.1 18.7

At the dose of 4 and 14 μM (doses confirmed as therapeuticallyeffective), peptide H14 is not toxic for normal hepatocytes.

2.3 Western Blot Results

NCI-H187 cells were incubated with concentrations of 1 μM and 10 μM ofpeptide H14. The response was measured at two incubation times (6 h and12 h). It was found that H14 induced apoptosis in the tested cell linesat both incubation times, increasing the amount of cleaved Parp proteinconsistently with the concentration (see FIG. 1).

NCI-H510A cells were incubated with concentrations of 1 μM and 10 μM ofpeptide-H14.

The response was measured after a 6 h incubation time. It was found thatH14 induced apoptosis in the tested cell line, consistently increasingthe amount of activated Caspases-3 with the concentration of peptide(see FIG. 2).

2.4. Stability Results

The experimental data are summarized in Table 11 below:

TABLE 11 H14 concentration and incubation H14 amount time (% peak area)Plasma, 100 ng/mL, 0 h, human 100 Plasma, 100 ng/mL, 1 h, human 96.1Plasma, 100 ng/mL, 2 h, human 96.1 Plasma, 100 ng/mL, 4 h, human 101.61Plasma, 100 ng/mL, 6 h, human 97.21 Plasma, 100 ng/mL, 24 h, human 108.5Plasma, 1000 ng/mL, 0 h, human 100 Plasma, 1000 ng/mL, 1 h, human 100.8Plasma, 1000 ng/mL, 2 h, human 94.9 Plasma, 1000 ng/mL, 4 h, human 95.4Plasma, 1000 ng/mL, 6 h, human 99.9 Plasma, 1000 ng/mL, 24 h, human100.6 *difference ±10% are considered not-significant according to themethod analysis

From the above data, it can be concluded that the peptides of theinvention have a high half-life and stability in human plasma.

2.5 Efficacy Results In Vivo

Tumor Growth

As can be seen in the Table 12, H14 reduced significantly tumor growthwith respect to the control. In particular, at the end-time (when thecontrol in sacrificed) the tumor reduction corresponds to 70%:

TABLE 12 Comparison of the tumor volume between groups of untreated mice(control) and treated mice with H14 i.p. Day of treatment Group 1 6 9 1621 Tumor Control 70.6 190.4 350.9 750.1 1150.3 Volume IDPH14 70.0 70.1150.1 250.4 350 25 mg/kg

As it is derived from these results, the administration of H14 peptideslows down cancer progression.

Thus, it can be concluded that H14 can be used as anticancer drug aloneor even in combination with chemo- or radiotherapy.

In view of the results provided above, the other peptides which havebeen in vitro tested, and which are active, are expected to work in thesame way as H14.

For reasons of completeness, various aspects of the invention are setout in the following numbered clauses:

Clause 1. A peptide of formula (I) or a pharmaceutical salt thereof:

wherein

“m”, “n”, “p”, and “q” represent integers and are selected from 0 and 1;and

“r” is comprised from 1 to 10;

the peptide comprising:

-   -   a linker biradical of formula (II)        —[(R₁)_(a)—(R₂)—(R₃)_(b)]_(c)—  (II)        which is connecting an alpha carbon atom of an amino acid        located at position “i” in the peptide sequence of formula (I)        with an alpha carbon atom of an amino acid located at position        “i+3” or “i+7” in the peptide sequence of formula (I),    -   a C-terminal end corresponding to —C(O)R₄; and    -   a N-terminal end corresponding to —NHR₅;        wherein    -   “a” and “b” are the same or different and are 0 or 1;    -   “c” is comprised from 1 to 10;    -   R₁ and R₃ are biradicals independently selected from the group        consisting of: (C₁-C₁₀)alkyl; (C₁-C₁₀)alkyl substituted by one        or more radicals selected from the group consisting of: halogen,        (C₁-C₁₀)alkyl, —OR₆, —NR₇R₈, —SR₉, —SOR₁₀, —SO₂R₁₁, and —CO₂R₁₂;        (C₂-C₁₀)alkenyl; (C₂-C₁₀)alkenyl substituted by one or more        radicals selected from the group consisting of: halogen,        (C₁-C₁₀)alkyl, —OR₆, —NR₇R₈, —SR₉, —SOR₁₀, —SO₂R₁₁, and —CO₂R₁₂;        (C₂-C₁₀)alkynyl; and (C₂-C₁₀)alkinyl substituted by one or more        radicals selected from the group consisting of: halogen,        (C₁-C₁₀)alkyl, —OR₆, —NR₇R₈, —SR₉, —SOR₁₀, —SO₂R₁₁, and —CO₂R₁₂;    -   R₂ is a biradical selected from the group consisting of: —O—,        C(═O), C(═O)NR₁₃, C(═O)O, S(═O), S(═O)₂, NR₁₄, (C₁-C₁₀)alkyl,        (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl, —NR₁₅—NR₁₆—, —N═N—, —S—S—, and        a known ring system comprising from 3 to 14 members, the system        comprising from 1 to 3 rings, where:        -   each one of the rings is saturated, partially unsaturated,            or aromatic;        -   the rings are isolated, partially or totally fused,        -   each one of the members forming the known ring system is            selected from the group consisting of: —CH—, —CH₂—, —NH—,            —N—, —SH—, —S—, and —O—; and        -   the ring system is optionally substituted by one or more            radicals independently selected from the group consisting of            halogen, —OH, —NO₂, (C₁-C₁₀)alkyl, (C₁-C₁₀)haloalkyl, and            (C₁-C₁₀)alkyl-O—; and    -   R₄ is a radical selected from the group consisting of —OH and        —NR₁₇R₁₈;    -   R₅ is a radical selected from the group consisting of —H and        (C₁-C₂₀)alkyl;    -   R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇ and R₁₈        are radicals independently selected from the group consisting        of: —H and (C₁-C₁₀)alkyl; and    -   the amino acids which are connected by the linker being of        formula (III)

-   -   wherein        -   R₁₉ is a monoradical selected from the group consisting of:            (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl, and a known            ring system comprising from 3 to 14 members, the system            comprising from 1 to 3 rings, where:        -   each one of the rings is saturated, partially unsaturated,            or aromatic;        -   the rings are isolated, partially or totally fused,        -   each one of the members forming the known ring system is            selected from the group consisting of: —CH—, —CH₂—, —NH—,            —N—, —SH—, —S—, and —O—.

Clause 2. The peptide of formula (I) or a pharmaceutical salt thereofaccording to clause 1, wherein “r”, “a”, “b”, and “c” are 1.

Clause 3. The peptide of formula (I) or a pharmaceutical salt thereofaccording to any one of the previous clauses, wherein R₁ and R₃ arebiradicals independently selected from the group consisting of:(C₁-C₁₀)alkyl; (C₂-C₁₀)alkenyl; and (C₂-C₁₀)alkynyl.

Clause 4. The peptide of formula (I) or a pharmaceutical salt thereofaccording to any one of the previous clauses, wherein R₂ is a biradicalselected from the group consisting of: (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl,and (C₂-C₁₀)alkynyl.

Clause 5. The peptide of formula (I) or a pharmaceutical salt thereofaccording to any one of the previous clauses, wherein R₁₉ is amonoradical selected from the group consisting of: (C₁-C₁₀)alkyl,(C₂-C₁₀)alkenyl, and (C₂-C₁₀)alkynyl.

Clause 6. The peptide of formula (I) or a pharmaceutical salt thereofaccording to any one of the previous clauses, wherein R₁, R₃ and R₁₉ are(C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; “m” and “n” are the same; and “p”and “q” are the same.

Clause 7. The peptide of formula (I) or a pharmaceutical salt thereofaccording to any one of the previous clauses, wherein the C-terminal endcorresponds to —C(O)OH or —C(O)NH₂, and the N-terminal end correspondsto —NH₂.

Clause 8. The peptide of formula (I) or a pharmaceutical salt thereofaccording to any one of the previous clauses, which is of formula (Ia)or (Ib):

wherein “m”, “n”, “p”, “q”, L, and R₁₉ are as defined above.

Clause 9. The peptide of formula (I) or a pharmaceutical salt thereofaccording to any one of the previous clauses, which is selected fromgroup consisting of SEQ ID NO: 2, 3, and 4.

Clause 10. The peptide of formula (I) or a pharmaceutical salt thereofaccording to any one of the previous clauses, which is conjugated to alabel or a drug.

Clause 11. A fusion protein comprising the peptide as defined in any oneof the previous clauses and, optionally, a cell penetrating peptide.

Clause 12. A pharmaceutical composition comprising a therapeuticallyeffective amount of the peptide or a pharmaceutical salt thereof asdefined in any one of the clauses 1-10, or the fusion protein as definedin clause 11, together with acceptable pharmaceutical excipients and/orcarriers.

Clause 13. A peptide or a pharmaceutical salt thereof as defined in anyone of the clauses 1-10 or a fusion protein as defined in clause 11 foruse as a medicament.

Clause 14. A peptide or a pharmaceutical salt thereof as defined in anyone of the clauses 1-10 or a fusion protein as defined in clause 11 foruse in the treatment of cancer.

Clause 15. The peptide or a pharmaceutical salt thereof for useaccording to clause 14, wherein the cancer is selected from the groupconsisting of: leukemia, breast cancer, glioblastoma, and lung cancer.

CITATION LIST

-   Altschul et al., “Basic local alignment search tool”, 1990, J. Mol.    Biol, v. 215, pages 403-410;-   Copolovici D. M. et al., “Cell-Penetrating Peptides: Design,    Synthesis, and Applications”, 2014, ACS Nano, 2014, 8 (3), pp    1972-1994;-   Higgins et al., “CLUSTAL V: improved software for multiple sequence    alignment”, 1992, CABIOS, 8(2), pages 189-191;-   Kim Young-Woo et al., “Synthesis of all-hydrocarbon stapled    a-helical peptides by ring-closing olefin metathesis”, Nature    Protocols, 2011, 6(6), p. 761-771;-   Kolb H. C. et al., “The growing impact of click chemistry on drug    discovery”, 2003, Drug Discov Today, 8(24):1128-1137); and-   Scott J. M. et al., “Application of Ring-Closing Metathesis to the    Synthesis of Rigidified Amino Acids and Peptides”, 1996, J. Am.    Chem. Soc., 1996, 118 (40), pp 9606-9614.

The invention claimed is:
 1. A peptide of formula (I) or apharmaceutical salt thereof:

wherein “m”, “n”, “p”, and “q” represent integers and are selected from0 and 1; and “r” is an integer from 1 to 10; the peptide comprising: alinker biradical “L” of formula (II)—[(R₁)_(a)—(R₂)—(R₃)_(b)]_(c)—  (II) which is connecting an alpha carbonatom of an amino acid located at position “i” in the peptide sequence offormula (I) with an alpha carbon atom of an amino acid located atposition “i+4” or “i+7” in the peptide sequence of formula (I), aC-terminal end corresponding to —C(O)R₄; and a N-terminal endcorresponding to —NHR₅; wherein, “a” and “b” are the same or differentand are 0 or 1; “c” is an integer from 1 to 10; R₁ and R₃ are biradicalsindependently selected from the group consisting of: (C₁-C₁₀)alkyl;(C₁-C₁₀)alkyl substituted by one or more radicals selected from thegroup consisting of: halogen, (C₁-C₁₀)alkyl, —OR₆, —NR₇R₈, —SR₉, —SOR₁₀,—SO₂R₁₁, and —CO₂R₁₂; (C₂-C₁₀)alkenyl; (C₂-C₁₀)alkenyl substituted byone or more radicals selected from the group consisting of: halogen,(C₁-C₁₀)alkyl, —OR₆, —NR₇R₈, —SR₉, —SOR₁₀, —SO₂R₁₁, and —CO₂R₁₂;(C₂-C₁₀)alkynyl; and (C₂-C₁₀)alkinyl substituted by one or more radicalsselected from the group consisting of: halogen, (C₁-C₁₀)alkyl, —OR₆,—NR₇R₈, —SR₉, —SOR₁₀, —SO₂R₁₁, and —CO₂R₁₂; R₂ is a biradical selectedfrom the group consisting of: —O—, C(═O), C(═O)NR₁₃, C(═O)O, S(═O),S(═O)₂, NR₁₄, (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl,—NR₁₅—NR₁₆—, —N═N—, —S—S—, and a known ring system comprising from 3 to14 members, the system comprising from 1 to 3 rings, where: each one ofthe rings is saturated, partially unsaturated, or aromatic; the ringsare isolated, partially or totally fused, each one of the membersforming the known ring system is selected from the group consisting of:—CH—, —CH₂—, —NH—, —N—, —SH—, —S—, and —O—; and the ring system isoptionally substituted by one or more radicals independently selectedfrom the group consisting of halogen, —OH, —NO₂, (C₁-C₁₀)alkyl,(C₁-C₁₀)haloalkyl, and (C₁-C₁₀)alkyl-O—; and R₄ is a radical selectedfrom the group consisting of —OH and —NR₁₇R₁₈; R₅ is a radical selectedfrom the group consisting of —H and (C₁-C₂₀)alkyl; R₆, R₇, R₈, R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇ and R₁₈ are radicals independentlyselected from the group consisting of: —H and (C₁-C₁₀)alkyl; and theamino acids of the peptide of formula (I) which are connected by thelinker biradical “L” are replaced by an amino acid of formula (III)

wherein R₁₉ is a monoradical selected from the group consisting of:(C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl, and a known ring systemcomprising from 3 to 14 members, the system comprising from 1 to 3rings, where: each one of the rings is saturated, partially unsaturated,or aromatic; the rings are isolated, partially or totally fused, eachone of the members forming the known ring system is selected from thegroup consisting of: —CH—, —CH₂—, —NH—, —N—, —SH—, —S—, and —O—; or,alternatively, a peptide or pharmaceutical acceptable salt thereofhaving an identity of at least 85% with the peptide of sequence SEQ IDNO: 18

wherein L is a linker biradical of formula (II) as defined above, and Xis an amino acid of formula (III), as defined above.
 2. The peptideaccording to claim 1 which is one of formula (I) or a pharmaceuticalsalt thereof:

wherein “m”, “n”, “p”, “q”, and “r” are as defined in claim 1, and thepeptide comprises a linker biradical of formula (II), a C-terminal endand a N-terminal end as defined in claim
 1. 3. The peptide or apharmaceutical salt thereof according to claim 1, wherein ‘r’, “a”, “b”,and “c” are
 1. 4. The peptide or a pharmaceutical salt thereof accordingto claim 1, wherein R₁ and R₃ are biradicals independently selected fromthe group consisting of: (C₁-C₁₀)alkyl; (C₂-C₁₀)alkenyl; and(C₂-C₁₀)alkynyl.
 5. The peptide or a pharmaceutical salt thereofaccording to claim 1, wherein R₂ is a biradical selected from the groupconsisting of: (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, and (C₂-C₁₀)alkynyl. 6.The peptide or a pharmaceutical salt thereof according to claim 1,wherein R₁₉ is a monoradical selected from the group consisting of:(C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, and (C₂-C₁₀)alkynyl.
 7. The peptide or apharmaceutical salt thereof according to claim 1, wherein R₁, R₃ and R₁₉are (C₁-C₁₀)alkyl; R₂ is (C₂-C₁₀)alkenyl; “m” and “n” are the same; and“p” and “q” are the same.
 8. The peptide or a pharmaceutical saltthereof according to claim 1, wherein the C-terminal end corresponds to—C(O)OH or —C(O)NH₂, and the N-terminal end corresponds to —NH₂.
 9. Thepeptide or a pharmaceutical salt thereof according to claim 1, which isof formula (Ia) or (Ib):

wherein “m”, “n”, “p”, “q”, L, and R₁₉ are as defined in any of thepreceding claims.
 10. The peptide or a pharmaceutical salt thereofaccording to claim 1, which is a peptide or pharmaceutical acceptablesalt thereof having an identity of at least 85% with the peptide ofsequence SEQ ID NO: 18 and it is selected from the group consisting of:(ibis1), (ibis2), (ibis3), (ibis4), (ibis5) and (ibis6):

wherein AA₃ is an amino acid other than Thr, and “X” and “L” are asdefined above;

wherein AA₄ is an amino acid other than Glu, and “X” and “L” are asdefined above;

wherein AA₅ is an amino acid other than Pro, and “X” and “L” are asdefined above;

wherein AA₂ is an amino acid other than Ala, AA₃ is an amino acid otherthan Thr, AA₄ is an amino acid other than Glu, and “X” and “L” are asdefined above;

wherein AA₁ is an amino acid other than Asn, and “X” and “L” are asdefined above;

wherein AA₂ is an amino acid other than Ala, and “X” and “L” are asdefined above.
 11. The peptide according to claim 1, which is selectedfrom the group consisting of SEQ ID NO: 2 to
 14. 12. The peptideaccording to claim 1, which is conjugated to a label, a drug or,alternatively, to a stabilizing moiety.
 13. The peptide according toclaim 12, which is of sequence SEQ ID NO:
 16. 14. A fusion proteincomprising the peptide or pharmaceutical acceptable salt thereof asdefined in claim 1 and, optionally, a cell penetrating peptide.
 15. Thefusion protein according to claim 14, which is of sequence SEQ ID NO:17.
 16. A pharmaceutical composition comprising a therapeuticallyeffective amount of the peptide or a pharmaceutical salt thereof ofclaim 1, together with acceptable pharmaceutical excipients and/orcarriers.
 17. A method of treating cancer, wherein the cancer isselected from the group consisting of leukemia, breast cancer,glioblastoma, and lung cancer, the method comprising administering atherapeutically effective amount of the peptide or a pharmaceutical saltthereof of claim 1 to a subject in need thereof.
 18. A method oftreating cancer, wherein the cancer is selected from the groupconsisting of leukemia, breast cancer, glioblastoma, and lung cancer,the method comprising administering a therapeutically effective amountof the fusion protein of claim 14 to a subject in need thereof.
 19. Amethod of treating cancer, wherein the cancer is selected from the groupconsisting of leukemia, breast cancer, glioblastoma, and lung cancer,the method comprising administering a therapeutically effective amountof the pharmaceutical composition of claim 16 to a subject in needthereof.
 20. The method of claim 17, wherein therapeutically effectiveamount of the peptide or a pharmaceutical salt thereof is administeredin combination with one or more anti-cancer agents.